Methods and compositions to treat myocardial conditions

ABSTRACT

Methods, devices, kits and compositions to treat a myocardial infarction. In one embodiment, the method includes the prevention of remodeling of the infarct zone of the ventricle using a combination of therapies. The method may include the introduction of structurally reinforcing agents. In other embodiments, agents may be introduced into a ventricle to increase compliance of the ventricle. The prevention of remodeling may include the prevention of thinning of the ventricular infarct zone. Another embodiment includes the reversing or prevention of ventricular remodeling with electro-stimulatory therapy. The unloading of the stressed myocardium over time effects reversal of undesirable ventricular remodeling. These therapies may be combined with structurally reinforcing therapies. In other embodiments, the structurally reinforcing component may be accompanied by other therapeutic agents. These agents may include but are not limited to pro-fibroblastic and angiogenic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/938,752, filed Nov. 12, 2007, U.S. Pat. No. 8,795,652, whichapplication is a divisional of U.S. patent application Ser. No.10/802,955, filed Mar. 16, 2004, U.S. Pat. No. 7,294,334, which is acontinuation-in-part of U.S. patent application Ser. No. 10/414,602,filed Apr. 15, 2003, U.S. Pat. No. 8,383,158.

FIELD OF THE INVENTION

The treatment of myocardial infarction, and more particularly, in oneembodiment, to the reinforcement of the infarct regional wall of a heartchamber using targeted cell delivery and/or the inhibition of thethinning of the infarct regional wall of a heart chamber using celldelivery in combination with other therapies such as electrostimulation.In further embodiments, to combine reinforcement of the infarct regionalwall and/or cellular replacement with stimulating the heart using apulsing and/or pacing device. In addition, this invention also pertainsto apparatus and methods for electrostimulation of the heart includingcardiac pacing with an artificial pacemaker to prevent or correct thenegative effects of remodeling. In particular, the invention relates tomethods for stimulating the heart in order to effect reversal ofmyocardial remodeling and provide structural support to the infarctregion.

BACKGROUND OF THE INVENTION

Ischemic heart disease typically results from an imbalance between themyocardial blood flow and the metabolic demand of the myocardium.Progressive atherosclerosis with increasing occlusion of coronaryarteries leads to a reduction in coronary blood flow. Blood flow can befurther decreased by additional events such as changes in circulationthat lead to hypoperfusion, vasospasm or thrombosis.

Myocardial infarction accounts for approximately 20% of all deaths. Itis a major cause of sudden death in adults.

Myocardial Infarction (MI) is one form of heart disease that oftenresults from the sudden lack of supply of oxygen and other nutrients.The lack of blood supply is a result of closure of the coronary arterythat nourishes the particular part of the heart muscle. The cause ofthis event is generally caused by arteriosclerosis “hardening of thearteries” in coronary vessels.

Formerly, it was believed that an MI was caused from a slow processionof closure from for example 95% then to100% but an MI can also be aresult of minor blockages where, for example, there is a rupture of thecholesterol plaque resulting in blood clotting within the artery. Thus,the flow of blood is blocked and downstream cellular damage occurs. Thisdamage can cause irregular rhythms that can be fatal, even though theremaining muscle is strong enough to pump a sufficient amount of blood.As a result of this insult to the heart tissue, scar tissue tends tonaturally form.

Even though relatively effective systemic drugs exist to treat MI suchas ACE-inhibitors and Beta-blockers, a significant portion of thepopulation that experiences a major MI ultimately develops heartfailure. An important component in the progression to heart failure isremodeling of the heart due to mechanical forces resulting in unevenstress and strain distribution in the left ventricle. Once an MI occursremodeling of the heart begins. The principal components of theremodeling event include myocyte death, edema and inflammation, followedby fibroblast infiltration and collagen deposition, and finally scarformation. The principle component of the scar is collagen. Since maturemyocytes of an adult are not regenerated the infarct region experiencessignificant thinning. Myocyte loss is the major etiologic factor of wallthinning and chamber dilation that may ultimately lead to progression ofcardiac myopathy. Myocyte death can and does occur. In other areas,remote regions experience hypertrophy (thickening) resulting in anoverall enlargement of the left ventricle. This is the end result of theremodeling cascade. These changes in the heart result in changes in thepatient's lifestyle and their ability to walk and to exercise. Thesechanges also correlate with physiological changes that result inincrease in blood pressure and worsening systolic and diastolicperformance.

FIGS. 1A-1C illustrate blood flow by longitudinal cross sectioning ofthe artery. FIG. 1A illustrates a normal unobstructed artery (artery100). FIG. 1B illustrates artery damage 110 due to a tear or spasm. Thisfigure illustrates a minor insult to the interior wall. FIG. 1Cillustrates an artery with plaque build-up 120 that reduces the bloodflow demonstrated by the blocked blood cell above the atheroscleroticmass. Fat and cholesterol build up at the site of damage. This mass canbe detected by methods currently available such as an, ECG, SPECT, MRI,and angiogram.

FIGS. 2A-2B illustrate the progression of heart damage once the build-upof plaque induces an infarct to occur. The most common pathogenesis ofthis disease is occlusive intracoronary thrombus where a thrombus iscovering an ulcerated stenotic plaque. This causes approximately 90% oftransmural acute myocardial infarctions. Other possible triggers of anMI are vasospasms with or without coronary atherosclerosis and possibleassociation with platelet aggregation. Another possible trigger isembolisms from left-sided mural thrombosis, vegetative endocarditis or aparadoxic embolism from the right side of the heart through a patentforamen ovale. FIG. 2A illustrates a site 210 in artery 200 whereblockage and restricted blood flow can occur from any of the indicatedcauses. FIG. 2B illustrates the extensive damage to the left ventriclethat can be a result of the lack of oxygen and nutrient flow carried bythe blood to the inferior region left ventricle of the heart. This area230 will likely undergo remodeling and eventually a scar will form and anon-functional (an area that does not contract) area will exist.

Significant atherosclerotic build-up can reduce the arterial lumen andreduce blood flow. Build-up is capable of rupturing resulting in a totalor partial occlusion of the artery. Complete coronary occlusion willlead to an acute MI. Thus the T-cells, platelets, fibrin and multipleother factors and cells are blocked from progression through the bloodstream and the result is an inadequate vascular supply as seen. Thisleads to myocyte death. Myocyte death, in addition to fibrosis in theform of collagen deposition, can lead to a compromised left ventricleand overload on the remaining myocytes. This process is furthercomplicated by compensation of the remaining myocytes that hypertrophy(enlarge). This can cause the left ventricle to enlarge and if the cyclecontinues can result in eventual heart failure.

The morphological appearance of the infarcted heart tissue post MI canvary. A transmural infarct involves the entire thickness of the leftventricular wall from the endocardium to the epicardium. It may extendinto the anterior free wall and the posterior free wall. This damage mayinclude extensions into the right ventricular wall. A subendocardialinfarct may have multiple focal regions and necrosis area may beconfined to the inner one-third to one-half of the left ventricularwall. The evolutionary changes in a subendocardial infarct do not evolvethe same as in a transmural MI.

Over time post-MI morphological changes occur. The gross morphologicalchanges that occur over approximately a 7-week period are pallor of themyocardium that leads to some hyperemia then yellowing central to thedamaged region. At approximately 15 days, the area is mostly yellow withsoft vascular margins. This area eventually turns white from fibrosis.On a microscopic level, the initial examination reveals wavy myocardialfibers. Coagulation and necrosis with loss of cross striations occurfollowed by contraction bands, edema, hemorrhage, and neutrophilicinfiltrate. Within 24-72 hours there is total loss of nuclei andstriations and heavy neutrophilic infiltrate. Then macrophage andmononuclear infiltration begin resulting in a fibrovascular response.Once this fibrovascular response occurs then prominent granulation ofthe tissue follows. This ultimately leads to fibrosis and a scar isformed by about 7 weeks post MI.

FIG. 3A-3B illustrate the occlusion of an artery that may lead to an MI.FIG. 3A illustrates the cross-section of a normal coronary artery withunobstructed lumen 301. The normal arterial wall 302 is made up of anintima layer 303, a media layer 304, and an adventitia layer 305. Withinthe arterial lumen, the intima is in direct contact with the flow ofblood. This region is mostly made up of endothelial cells. The medialayer is mostly smooth muscle cells and extracellular matrix proteins.Finally, the adventitia layer is primarily made up of collagen, nerves,blood vessels and lymph vessels. FIG. 3B illustrates a coronary arterywith atherosclerosis. In this example, this artery is about 50 percentoccluded (only 50 percent of the arterial lumen is free of obstruction).Thus, the obstructed artery may lead to damage observed in a ventricleof an MI subject.

After an MI has occurred, three layers of tissue can be distinguished.The infarct region has (1) the region of significant necrosis/apoptosistissue (2) the border zone that consists of a large concentration ofapoptotic and necrotic tissue as well as viable tissue and (3) theunaffected region that consists of mainly viable tissue. In the borderzone the cells exist in an oxygen-deprived state due to the damage fromthe MI.

FIG. 3C-3J illustrate the details of a post-MI remodeling of theventricle. The progression of heart failure after an MI is a result ofthe remodeling of the heart after the infarct. The remodeling processcauses infarcted region of the heart to stretch and become thinnercausing the left ventricular diameter to increase. As the heartcontinues to remodel, the stresses on the heart increase. FIG. 3C, on acellular level, a normal myocardium is illustrated. FIG. 3C illustratesthe cross striations 306 and central nuclei 307 of a healthy myocytepopulation.

FIG. 3D-3J depict the progression of the remodeling of the ventriclepost MI. FIG. 3D illustrates an early acute MI. Here, there areprominent pink contraction bands that are indicated by reference number308. FIG. 3E illustrates the increasing loss of striations and somecontraction bands. The nuclei in this illustration are incurringkaryolysis, i.e., a stage of cell death that involves fragmentation of acell nucleus; the nucleus breaks down into small dark beads of damagedchromatin 309. In addition, the neutrophils are infiltrating the damagedmyocardial region. FIG. 3F illustrates an acute MI. The loss of nucleiand loss of cross striations are evident. There is extensivehemorrhaging on the infarct border 310. FIG. 3G illustrates theprominent necrosis and hemorrhaging 310, as well as the neutrophilicinfiltrate 311. Subsequently, a yellowish center is formed within thedamaged area with necrosis and inflammation surrounded by the hyperemicborder. After 3-5 days post-MI, the necrosis and inflammation areextensive. There is a possibility of rupture at this point. FIG. 3Hillustrates approximately one week after the MI with capillaries,fibroblasts and macrophages filled with haemosiderin (haemosiderin is along-term reserve (storage form) of iron in tissues) 312. In two tothree weeks granulation is the most prominent feature observed. FIG. 3Iillustrates extensive collagen deposition 313 seen after a couple ofweeks. Collagenous scarring occurs in subendocardial locations in remotemyocardial infarct regions. FIG. 3J illustrates the myocytes 314 afterseveral weeks of healing post MI. They are hypertrophied with large darknuclei 315 and interstitial fibrosis 316. These enlarged cellscontribute to the enlarged left ventricle.

A complication of an MI is an aneurysm that looks like a bulge in theleft ventricular wall. The aneurysm is made up of non-functional tissuethat is unable to contract. Therefore, the ejection and stroke volume ofthe heart are reduced. Additionally, parts of this mass can form a muralthrombus that can break off and embolize to the systemic circulation.

Heart Stimulation and the Use of Prostaglandins

The body essentially produces two types of prostaglandins; “good”prostaglandins and “bad” prostaglandins. Prostaglandins are hormone-likesubstances that regulate many body processes, such as blood clotting.“Good” prostaglandins (PG1 and PG3) regulate heart function, improveblood flow and prevent platelets from sticking together. A diet rich inOmega-3 fatty acids leads to the production of PG3, which is beneficial.

Prostaglandins are compounds that are produced via the metabolism offats in our diets. These compounds are simplistically categorized aseither “good” or “bad.” The good prostaglandins are beneficial andconstructive to the body while the bad ones, if produced on a continualbasis, can be destructive.

Prostaglandins are hormone-like substances, which have wide andsignificant effects in regulating many vital life processes. Theimportance of the role of these compounds has been appreciated only inthe last decade. One type of prostaglandin E prevents platelets—a bloodconstituent—from clotting. This discovery may be applied clinicallyagainst heart attacks and strokes caused by clots. These prostaglandins,by inhibiting the secretion of gastric acid in the stomach, may also byuseful in the treatment of gastric ulcer.

The E type prostaglandin, a powerful dilator of blood vessels, has beenfound in animal experiments to reduce high blood pressure—another causeof heart attacks and stroke. Such blood pressure reduction appears to bethe result of accelerated water excretion and inhibition of sodiumretention.

In one study, prostaglandin E2 (PGE2) was used to maintain patency ofthe ductus arteriosus in four neonates with cyanotic congenital heartdisease due to obstructive right heart malformations. PGE2 was infusedprior to surgery, and in three patients, during surgery until asatisfactory aortopulmonary shunt was established. PGE2 producedconsistently an immediate and persistent rise in arterial oxygensaturation, which could be ascribed to dilation of the ductusarteriosus. No major side effects occurred, except for pyrexia in twoinfants. All patients recovered well from surgery. This treatment wasproposed as a treatment for preparation for surgery in any infant withcongenital heart defects and ductus-dependent pulmonary blood flow. Thesame treatment may be useful preoperatively in patients with aorticinterruption who also depend on continued patency of the ductus forblood supply to the lower half of the body

Pacing and Pulse Generators (PG)

An implantable pacemaker pulse generator is a device that has a powersupply and electronic circuits that produce a periodic electrical pulseto stimulate the heart. This device is used as a substitute for theheart's intrinsic pacing system to correct both intermittent andcontinuous cardiac rhythm disorders. This device includes triggered,inhibited, and asynchronous devices implanted in the human body.

Electrical stimulation of the heart underlies cardiac pacing anddefibrillation. The “bidomain model” describes the anisotropicelectrical properties of cardiac tissue. In particular, this modelpredicts mechanisms by which applied electric fields change thetransmembrane potential of the myocardial cells. During unipolarstimulation, the bidomain model can explain “make” and “break”stimulation. Furthermore, it elucidates the cause of the “dip” in theanodal strength-interval curve, and predicts the initiation of novelquatrefoil reentry patterns. These results are beginning to shed lighton the mechanisms of arrhythmia induction and defibrillation.

What is needed is to prevent thinning of the infarct region, replacedead cells with viable cells stimulation of the removal/replacement ofthe tissue affected by myocardial infarction to enhance the ECMproduction.

SUMMARY

Embodiments herein relate to methods, apparati and compositions forreversing ventricular remodeling using electro-stimulatory therapy incombination with other methods such as structural reinforcement of thearea to prevent remodeling and strengthen an infarct region. By pacingsites in proximity to the infarct with appropriately timed pacingpulses, the infarct region is pre-excited in a manner that lessens themechanical stress to which it is subjected, thus reducing the stimulusfor adverse remodeling. Reducing the wall stress of the infarct regionalso decreases the probability of an arrhythmia arising from the region.Another advantage obtained with resynchronizing the ventricularcontraction by pre-exciting a weakened infarct region is the creation ofhemodynamically more efficient contraction.

In one embodiment, the ventricular stimulatory pulse or pulses may bedelivered in accordance with a programmed bradycardia pacing mode inresponse to sensed cardiac activity and lapsed time intervals. Inanother embodiment, a stimulating/sensing electrode is disposed in theventricle at a selected site in proximity to a stressed region. Pacingthat pre-excites the ventricle at this site results in the stressedregion being excited before other regions of the ventricular myocardiumas the wave of excitation spreads from the paced site. Other embodimentsinvolve multi-site pacing in which multiple stimulating/sensingelectrodes are disposed in the ventricles. Pacing the ventricles duringa cardiac cycle then involves outputting pulses to the electrodes in aspecified sequence. In one embodiment, the pulse output sequence may bespecified such that a stressed region of the ventricular myocardium isexcited before other regions as the wave of excitation spreads from themultiple pacing sites.

In one embodiment, a composition is described that is capable ofreplacing the myocardial cells and may provide reinforcement to theventricle in addition to the pacing methods describe previously. Thereplacement cells consist of cells that do not trigger an immuneresponse often seen in graft rejection. In another embodiment, a methodis described to increase the compliance of a ventricle by preventingthinning of the infarct region and eliminating the cellular debriscreated by the infarct. A cellular bolus may be advanced through adelivery device to the infarct zone for reinforcement. In oneembodiment, the cellular therapy may be combined with stimulatorytherapies created by electrical and/or chemical stimuli. In otherembodiments, a treatment agent is delivered via multiple small volumesto the region. These delivery methods may use imaging of the ventricularwall to guide the deposition of the treatment agent to the site of theinfarct zone such as deposition of the gel-forming agents.

In one embodiment, the cells delivered to the infarct region maycomprise an immunotolerant or non-antigenic cell line such as, but notlimited to, α-1,3-galactosyltransferase (GGTA1) knockout swine cells. Inother embodiments, treatment agents may also be used to induceangiogenesis.

In another embodiment, one or more components may be delivered bymicroparticles harboring the component and/or therapeutic agents orgrowth factors. Cells that infiltrate the infarct region may bestimulated to proliferate. In other embodiments, cellular treatments maybe combined with a peri-infarct treatment such as electrical stimuli byleads in contact with the infarct region that encourages new growth inthe infarct area. This modulation of tissue response in the infarct zoneis suitable for reinforcing the region and preventing the thinningprocess of the ventricular wall.

Other embodiments directed to the prevention of thinning of the infarctregion of the ventricle wall are included. Treatment agents that arecapable of cross-linking the existing collagen in the infarct region aredescribed. The cross-linked collagen would form a structurallyreinforcing wall in the infarct region to bulk-up the infarct zone andreduce the effects of thinning. This may be combined with the cellularreplacement and/or electro-stimulatory therapy.

In another embodiment, a method includes multi-component treatments ofthe infarct zone. One multi-component method includes the formation of ascaffold to facilitate the attachment of cells and to deliver growthfactors and other treatment agents. In addition, the in-growth of newcapillaries is encouraged by the sustained release of angiogenic factorsby the microparticles that form the scaffold. The treatment agents maybe released for up to two months period. This technique would offermaximum benefit for the regeneration of viable tissue. These treatmentsmay be used prior to, in conjunction with or after electro-stimulatorytherapy.

In another embodiment, a different multi-component treatment of theinfarct zone introduces a scaffold system that provides a matrix tofacilitate cell growth and attenuate the remodeling event post-MI. Inaddition, the treatment may include a perfluorinated compound thatenhances the re-oxygenation of the tissue. These methods may also becombined with electro-stimulatory therapies in order to enhance therecovery period of a subject (e.g., a subject with heart disease).

In one embodiment, the treatments proposed may occur at any time after aheart defect such as but not limited to an infarction or heart failure.In another embodiment, the treatments proposed may occur within sevenweeks of an MI event (or prior to myocyte replacement). In anotherembodiment, the treatments proposed may occur within two weeks of an MIevent.

In a further embodiment, a kit is disclosed. One example of such a kitis a kit including an injectable cell composition that may be introducedto the infarct region directly. Optionally, a kit may contain a pacegenerating device or other stimulatory device.

BRIEF DESCRIPTION OF THE DRAWINGS

The methods and compositions are illustrated by way of example, and notlimitation, in the figure of the accompanying drawings in which:

FIG. 1A illustrates a longitudinally sectioned healthy artery and bloodflow therein.

FIG. 1B illustrates a longitudinally sectioned damaged artery due to atear or a spasm.

FIG. 1C illustrates a longitudinally sectioned occluded artery due tofat and cholesterol build up.

FIG. 2A illustrates plaque build up in an artery that may result inrestriction of blood and oxygen flow to the heart.

FIG. 2B illustrates the damage to the heart as a result of the plaquebuild-up in an artery that lead to an MI.

FIG. 3A illustrates a normal artery.

FIG. 3B illustrates an artery with arteriosclerosis (50 percentblockage) that may lead to an MI.

FIG. 3C illustrates normal myocardium.

FIG. 3D illustrates an example of myocardium of an early acutemyocardial infarction.

FIG. 3E illustrates an example of myocardium of an early myocardialinfarction whereby a myocardium demonstrates increasing loss of crossstriations.

FIG. 3F illustrates an example of myocardium of an acute myocardialinfarction and the loss of striations and the nuclei.

FIG. 3G illustrates an example of myocardium of an acute myocardialinfarction resulting in neutrophilic infiltration and necrosis.

FIG. 3H illustrates an example of the myocardium of an acute myocardialinfarction approximately one week after a myocardial infarctionoccurred. The capillaries, fibroblasts and macrophages fill withhemosiderin.

FIG. 3I illustrates an example of the myocardium a couple of weeks aftera myocardial infarction. A lot of collagen has been deposited at thesite of damage.

FIG. 3J illustrates myocardium several weeks after a myocardialinfarction. Many surviving myocytes appear hypertrophic and their nucleiare dark in color. Interstitial fibrosis is also observed.

FIG. 4 illustrates an exemplary flow chart of the introduction of acellular composition to an infarct zone and the replacement of the deadtissue.

FIGS. 5A-53 illustrate a multi-component method for electrostimulatingand structurally reinforcing an infarct region.

FIG. 6 illustrates an exemplary flow chart of stimulation of the heartcombined with cellular replacement therapy in the infarct region.

FIG. 7 illustrates a flowchart of introducing cellular compositions tothe infarct region and stimulating the region with leads.

FIG. 8 illustrates a general structure of a first and of a secondcomponent that may form a scaffold-like structure for reinforcement inan infarct region.

FIGS. 9A-9E illustrates introduction and action of the methodsillustrated in the flowchart of FIG. 7 in an infarct region.

DEFINITIONS

“a container”—a receptacle, such as a carton, can, vial, tube, bottle,or jar, in which material is held or carried.

“cardiomyocyte-like”—a cell(s) capable of converting to acardiomyocyte(s) or a cell or components capable of functioning like acardiomyocyte and/or expressing one or more cardiac specific molecularmarker(s).

“polymer-forming”—any agent or agents capable of forming a gelatinousmaterial either alone or in combination.

“delivery device”—an apparatus or system capable of depositing asolution, powder, concentrate, a single reagent and/or multiplereagents.

“pro-fibroblastic” agent—one or more compounds capable of retaining,inducing proliferation of and/or recruiting fibroblasts cells.

“compliance”—The ability of a blood vessel or a cardiac chamber tochange its volume in response to changes in pressure has importantphysiological implications. In physical terms, the relationship betweena change in volume (D V) and a change in pressure (D P) is termedcompliance (C), where C=ΔV/ΔP. Compliance, therefore, is related to theease by which a given change in pressure causes a change in volume. Inbiological tissues, the relationship between ΔV and ΔP is not linear.Compliance is the slope of the line relating volume and pressure thatdecreases at higher volumes and pressures. Another way to view this isthat the “stiffness” of the chamber or vessel wall increases at highervolumes and pressures. Changes in compliance have importantphysiological effects in cardiac chambers and blood vessels.

DETAILED DESCRIPTION

In the following section, several embodiments of, for example,processes, compositions, devices and methods are described in order tothoroughly detail various embodiments. It will be obvious though, to oneskilled in the art that practicing the various embodiments does notrequire the employment of all or even some of the specific detailsoutlined herein. In some cases, well known methods or components havenot been included in the description in order to prevent unnecessarilymasking various embodiments.

Methods and compositions to treat a ventricle after a myocardialinfarction (MI) are disclosed. In one embodiment, the infarct region orthe area of the ventricle containing the infarct injury may be treatedalone or in combination with other treatments. One benefit to suchtreatment is that the region of injury may be targeted with little or noaffect on the outlying healthy heart tissue. In addition, anotherbenefit of such treatment is that the treatment may prevent the loss offunctionality of a region of injury due to the normal remodeling andscar forming procedure to mend an infarct region. Another benefit may bethat the treatment may increase the compliance of the ventricle. Anotherbenefit may be that the treatment may reduce or eliminate the debrisfound in the post infarct region to encourage an angiogenic responseand/or allow/enhance the microenvironment for ECM scaffoldremodeling/regeneration. Still another benefit is the reduction inthinning of a ventricular wall of an infarct zone. In the followingdescription, structural reinforcement of the infarct region of theventricle is described. Since most myocardial infarctions occur in theleft ventricle most descriptions will be directed towards left ventriclerepair. But, it is appreciated that treatment of the right ventricle maybe achieved in a similar manner.

If the remodeling of the infarct region could be modified prior to scarformation and ultimate thinning of the ventricular wall, functionaltissue may be rescued. The inhibition of scar formation and guidedregeneration of viable cells would lead to increased wall strength andalter collagen deposition, instead of thinning and hypertrophiedmyocytes. Further, decreasing the probability of wall thinning andfortifying the influx of cellular components such as immunotolerantcells might be beneficial and preferred over the current treatment of anMI, namely continual exposure to systemic drugs to treat the symptomsand not the disease. Another benefit may be that any one of thetreatments herein may result in an increase in compliance of theventricle. Thus, any one or more combinations of these treatments mayprovide a potential for healing the infarct region and prevention offurther complications.

In other embodiments, a kit (e.g., a pre-manufactured package) isdisclosed. A suitable kit includes at least one component (e.g.,cellular component such as α-1,3-galactosyltransferase (GGTA1) knockoutheart cells or cardiomyocyte-like cell(s) capable of converting to acardiomyocyte(s) or a cell or components capable of functioning like acardiomyocyte) and/or agent and a lumen to house the component. Thecomponent has a property that may increase the modulus (tensilestrength, “stiffness”) of elasticity of the infarct region, increasecompliance of the ventricle and/or prevent or reduce thinning caused byremodeling (e.g., by replacing the myocardial cells). The kit may besuitable, in one example, in the methods described.

Electrical System of the Heart

When a transmural myocardial infarction in the left ventricle occurs,the affected area suffers a loss of contractile fibers that depends uponthe degree of collateral circulation to the area. For example, theinfarction may either leave a non-contractile scar or leave some viablemyocardium interspersed with scar tissue, with the myocardial fibersthat surround the infarcted area suffering a variable amount ofdestruction. In any case, regions in and around the infarct sufferimpaired contractility, and this is responsible for the ventriculardysfunction that initiates the remodeling process as described above.Whether the infarction results in a non-contractile scar or a fibrousregion with diminished contractility, the viable myocardium in proximityto the infarct are the regions of the ventricle that are least able torespond to the increased stresses brought about by ventriculardysfunction in a physiologically appropriate manner. These regions arethus the parts of the ventricle that are most vulnerable to thepost-infarct remodeling process. In one embodiment, a method to treat inthe proximity of the infarct to lessen mechanical stress withoutadversely compromising ventricular systolic function and decrease theundesirable remodeling of the region is proposed. Known and conventionalelectrostimulatory techniques may be used in the context of any of theembodiments.

The degree to which a heart muscle fiber is stretched before itcontracts is termed the preload, while the degree of tension or stresson a heart muscle fiber as it contracts is termed the afterload. Themaximum tension and velocity of shortening of a muscle fiber increaseswith increasing preload, and the increase in contractile response of theheart with increasing preload is known as the Frank-Starling principle.When a myocardial region contracts late relative to other regions, thecontraction of those other regions stretches the later contractingregion and increases its preloading, thus causing an increase in thecontractile force generated by the region. Conversely, a myocardialregion that contracts earlier relative to other regions experiencesdecreased preloading and generates less contractile force. Becausepressure within the ventricles rises rapidly from a diastolic to asystolic value as blood is pumped out into the aorta and pulmonaryarteries, the parts of the ventricles that contract earlier duringsystole do so against a lower afterload than do parts of the ventriclescontracting later. Thus, one embodiment proposes to treat a ventricularregion to induce contraction earlier than parts of the ventricle todecrease both the preload and afterload which may decrease themechanical stress experienced by the region relative to other regions.This may result in the region being more efficient by lessening itsmetabolic demands such as oxygen requirements.

In one embodiment, electrostimulatory pacing pulses may be delivered toone or more sites in or around the infarct in a manner that pre-excitesthose sites relative to the rest of the ventricle. As the term is usedherein, a pacing pulse is any electrical stimulation and possiblytargeted chemical stimulation of the heart of sufficient energy toinitiate a propagating depolarization, whether or not intended toenforce a particular heart rate. In a normal heartbeat, the specializedHis-Purkinje conduction network of the heart rapidly conducts excitatoryimpulses from the sinoatrial node to the atrio-ventricular node thatlikely results in a coordinated contraction of both ventricles.

Artificial pacing with an electrode fixed into an area of the myocardiumdoes not take advantage of the heart's normal specialized conductionsystem for conducting excitation throughout the ventricles because thespecialized conduction system can only be entered by impulses emanatingfrom the atrio-ventricular node. Thus, the spread of excitation from aventricular pacing site must proceed only via the much slower conductingventricular muscle fibers, resulting in the part of the ventricularmyocardium stimulated by the pacing electrode contracting well beforeparts of the ventricle located more distally to the electrode. Thispre-excitation of a paced site relative to other sites can be used todeliberately change the distribution of wall stress experienced by theventricle during the cardiac pumping cycle. Pre-excitation of theinfarct region relative to other regions unloads the infarct region frommechanical stress by decreasing its afterload and preload, thuspreventing or minimizing the remodeling that would otherwise occur.Since the contractility of the infarct region is impaired,pre-excitation of the region may result in a resynchronized ventricularcontraction that is hemodynamically more effective. This may bebeneficial in reducing the stimulus for remodeling and reducing theincidence of angina due to coronary insufficiency. Decreasing the wallstress of the infarct region also reduces its oxygen requirements anddecreases the probability of an arrhythmia arising in the region.

In one embodiment, pacing therapy to unload the infarct region may beimplemented by pacing the ventricles at a single site in proximity tothe infarct region or at multiple ventricular sites. With multiplesites, the pacing pulses may be delivered simultaneously or in acontrolled pulse output sequence. As outlined below, the single-site ormultiple site pacing may be performed using a bradycardia pacingalgorithm such as an inhibited demand mode or a triggered mode.

In one embodiment, to pre-excite the infarct region, one or more pacingelectrodes may be placed in proximity to the region. To locate theinfarct region several techniques to map the heart may be usedincluding, but not limited, to ultrasonic imaging, PET scans, andthallium scans, and MRI perfusion scans. In the case of a leftventricular infarct, epicardial leads may be placed directly on theepicardium with a thoracotomy (an open chest surgical operation) or witha thorascopic procedure, or leads may be threaded from the upper venoussystem into a cardiac vein via the coronary sinus. (See, e.g., U.S. Pat.No. 5,935,160 incorporated herein by reference.)

Pacemakers

A permanent pacemaker is a small device that is implanted under the skin(often in the shoulder area just under the collarbone), and it sendselectrical signals to start or regulate a slow heartbeat. As the term isused herein, a “pacemaker” should be taken to mean any cardiac rhythmmanagement device with a pacing functionality regardless of any otherfunctions it may perform. A permanent pacemaker may be used to make theheartbeat if the heart's natural pacemaker (the sinoatrial node SA node)is not functioning properly and has developed an abnormal heart rate orrhythm, or if the electrical pathways are blocked.

Pacemakers are usually implanted subcutaneously on the patient's chest,and are connected to sensing/pacing electrodes by leads either threadedthrough the vessels of the upper venous system to the heart or by leadsthat penetrate the chest wall. The controller of the pacemaker may bemade up of a microprocessor communicating with a memory via abidirectional data bus, where the memory typically comprises a ROM(read-only memory) for program storage and a RAM (random-access memory)for data storage. The controller may be implemented by other types oflogic circuitry (e.g., discrete components or programmable logic arrays)using a microprocessor-based system. The controller is capable ofoperating the pacemaker in a number of programmed modes where aprogrammed mode defines how pacing pulses are output in response tosensed events and expiration of time intervals. A telemetry interfacemay also be provided for communicating with an external programmer.

An electrical stimulus is generated by the sinus node (also called thesinoatrial node, or SA node), which is a small mass of specializedtissue located in the right atrium (right upper chamber) of the heart.The sinus node generates an electrical stimulus periodically. Thiselectrical stimulus travels down through the conduction pathways andcauses the heart's chambers to contract and pump out blood. The rightand left atria (the two upper chambers of the heart) are stimulatedfirst and contract a short period of time before the right and leftventricles (the two lower chambers of the heart). The electrical impulsetravels from the sinus node to the atrioventricular (AV) node, where itstops for a very short period, then continues down the conductionpathways via the bundle of His into the ventricles. The bundle of Hisdivides into right and left pathways to provide electrical stimulationto both ventricles.

Normally, as the electrical impulse moves through the heart, the heartcontracts. One contraction represents one heartbeat. The atria contracta fraction of a second before the ventricles so their blood empties intothe ventricles before the ventricles contract.

Under some conditions, almost all heart tissue is capable of starting aheartbeat, or becoming the pacemaker. Some symptoms of arrhythmiasinclude, but are not limited to, weakness, fatigue, palpitations, lowblood pressure, and dizziness and fainting. The symptoms of arrhythmiasmay resemble other conditions or medical problems.

Components of a Permanent Pacemaker/ICD

In one embodiment, a permanent pacemaker may be used. A permanentpacemaker has three principle components a pulse generator, a sealedlithium battery and an electronic circuitry package. The pulse generatorproduces the electrical signals that make the heart beat. Many pulsegenerators also have the capability to receive and respond to signalsthat are sent by the heart itself. One or two wires (also called leads)are insulated flexible wires that conduct electrical signals to theheart from the pulse generator. The leads may also relay signals fromthe heart to the pulse generator. One end of the lead is attached to thepulse generator and the electrode end of the lead is positioned in theatrium (the upper chamber of the heart) or in the ventricle (the lowerchamber of the heart). Pacemaker technology is now much more advanced.Today, pacemakers are able to “sense” when the heart's natural ratefalls below the rate that has been programmed into the pacemaker'scircuitry. Pacemaker leads may be positioned in the atrium or ventricle,infarct area or any combination of sites depending on the conditionrequiring the pacemaker to be inserted. Atrial pacemakers andventricular pacemakers are well known in the art. It is possible to havea pacemaker with leads in any, all or a combination of sites.

In addition, specialty pacemakers that pace either the right atrium orthe right ventricle are called “single-chamber” pacemakers. Pacemakersthat pace both the right atrium and right ventricle of the heart andrequire two pacing leads are called “dual-chamber” pacemakers.

Implanting a Pacemaker

In one embodiment, a Pacemaker/ICD insertion may be performed as anoutpatient procedure, done in the cardiac catheterization laboratory, orthe electrophysiology laboratory. The patient may be awake during theprocedure, although sedation may be given to help the patient relaxduring the procedure. A small incision may be made just under thecollarbone. The pacemaker/ICD lead(s) may be inserted into the heartthrough a blood vessel that runs under the collarbone. Once the lead isin place, it is tested to make sure it is in the right place and isfunctional. The lead may then be attached to the generator, which may beplaced just under the skin through the incision made earlier.

Pre-Excitation Pacing

In one embodiment, pre-excitation pacing may be applied to one or moreventricular sites in proximity or within an infarct region which may bedelivered by a bradycardia pacing mode, which refers to a pacingalgorithm that enforces a certain minimum heart rate. Pacemakers mayenforce a minimum heart rate either asynchronously or synchronously. Inasynchronous pacing, the heart may be paced at a fixed rate regardlessof an intrinsic cardiac activity. Because of the risk of inducing anarrhythmia with asynchronous pacing, most pacemakers for treatingbradycardia may be programmed to operate synchronously in a “demandmode” where sensed cardiac events occurring within a defined intervaleither trigger or inhibit a pacing pulse. Inhibited demand pacing modesutilize escape intervals to control pacing in accordance with sensedintrinsic activity. In an inhibited demand ventricular pacing mode, theventricle may be paced during a cardiac cycle only after expiration of adefined escape interval during which no intrinsic beat by the chamber isdetected. For example, a ventricular escape interval may be definedbetween ventricular events so as to be restarted with each ventricularsense or pace. The inverse of this escape interval is the minimum rateat which the pacemaker will allow the ventricles to beat, sometimesreferred to as the lower rate limit (LRL). In an atrial tracking pacingmode, another ventricular escape interval may be defined between atrialand ventricular events, referred to as the atrio-ventricular interval(AVI). The atrio-ventricular interval is triggered by an atrial senseand stopped by a ventricular sense or pace. A ventricular pace isdelivered upon expiration of the atrio-ventricular interval if noventricular sense occurs before the expiration. It may be desirable incertain subjects (e.g., humans) to decrease the AVI to be below theintrinsic PR interval (i.e., the normal time for an intrinsicventricular beat to occur after an atrial sense) or increase the LRL tobe slightly above the patient's normal resting heart rate. In otherembodiments, pre-excitation pacing therapy may be started, stopped, ormodified based upon sensor measurements. For example, the pacemakercould measure the impedance between pairs of electrodes to detect wallmotion or changes in wall thickness during the cardiac cycle. Separatepairs of electrodes can be used to produce impedance signals from both apaced region and a non-ischemic region, such as the right ventricle ifthe paced and ischemic region is in the left ventricle. Ischemia in thepaced region may be monitored by comparing the timing of the contractionin the paced region with the timing of the non-ischemic region. If thecontractions in the paced region are delayed or significantly prolonged,an increase in ischemia can be surmised, and pre-excitation pacing tothe area can either be started or increased. Conversely, if a decreasein ischemia is detected, pre-excitation pacing may either be stopped orreduced. Modifications to the pacing therapy can also be made inaccordance with detected changes in the wall thickness of the pacedregion. In another embodiment, an accelerometer or microphone on thepacing lead or in the device package may be used to sense the acousticenergy generated by the heart during a cardiac cycle. Changes in theamplitude or morphology of the acoustic energy signal may then be usedto infer changes in the wall motion and the efficiency of contractionand relaxation. The applied pre-excitation pacing therapy may then bemodified based upon this information. (See U.S. Pat. No. 6,058,329 andU.S. Pat. No. 6,628,988, hereby incorporated by reference.) A device fordelivering pre-excitation pacing therapy as described above may alsohave other functionality that can be of benefit to patients withischemic heart disease, such as cardioversion/defibrillation. In oneembodiment, a device for delivering pre-excitation pacing therapy asdescribed may be combined with any reinforcement and/or cellularreplacement composition.

Pacing Units

In one embodiment, an implanted cardiac rhythm management device mayautomatically adjust the pulse output sequence in accordance withmeasurements of myocardial mass. Such measurements may be made bymeasuring the conduction delays of excitation spreading through themyocardium as sensed by multiple sensing/stimulation electrodes.Increased conductions delays through a region, for example, may bereflective of stress in the region that can be reduced by pre-excitationstimulation. In another embodiment, impedance measurements may be madebetween electrodes in proximity to the heart that correlate withvariations in myocardial mass and contraction sequence. Suchmeasurements may be used to identify akinetic or dyskinetic regions ofthe myocardium as well as to indicate wall thickness abnormalities. Theparticular pre-excitation interval used by the device may also beautomatically adjusted in accordance with detected changes in theremodeling process. That is, the pre-excitation interval may beshortened as remodeling is reversed, or increased as remodeling worsens.Remodeling changes can be detected by, for example, measuring changes ortrends in conduction delays, contraction sequences, end-diastolicvolume, stroke volume, ejection fraction, wall thickness, or pressuremeasurements.

In another embodiment, the pulse output sequence used by a cardiacrhythm management may be alternated between one designed to producehemodynamically more effective contractions when metabolic needs of thebody are great to one designed to promote reverse remodeling whenmetabolic needs are less. A pulse output sequence that unloads ahypertrophic region may not be the optimum pulse output sequence formaximizing hemodynamic performance. For example, a more hemodynamicallyeffective contraction may be obtained by exciting all areas of themyocardium simultaneously, which may not effectively promote reversal ofthe hypertrophy or remodeling. The pulse output sequence may thereforebe adjusted automatically in accordance with exertion level measurementsreflective of metabolic demand so that pulse output sequences thatunload hypertrophied or stressed regions are not used during periods ofincreased exertion.

Mapping of the Heart

In each of the methods described herein, it is appreciated that specificareas of the heart may be targeted for application of any of theincorporated methods, thus there are techniques previously describedthat may be used for targeting the infarct region. One example oftargeting a specific region such as an infarct zone uses a techniqueknown as mapping the heart (U.S. Pat. No. 6,447,504). The data areacquired by using one or more catheters that are advanced into theheart. These catheters usually have electrical and location sensors intheir distal tips. Some of the catheters have multiple electrodes on athree-dimensional structure and others have multiple electrodesdistributed over a surface area. One example of the later catheter maybe a sensor electrode distributed on a series of circumferences of thedistal end portion, lying in planes spaced from each other. Thesetechniques provide methods to characterize the condition of the heart insome situations using electrical potentials in the heart tissue as wellas using electromechanical mapping, ultrasonic mapping to map the viableand the non-viable regions of the heart for example the left ventricleand the infarct zone. In addition, the ultrasound waves may be used todetermine the thickness of the heart tissue in the vicinity of the probefor example, sensing the characteristic of the heart tissue by analyzingthe ultrasound signals to determine the depth of the channels. Anothermethod known as viability mapping (for example Spect, MRI, PET) may alsobe used. Viability mapping may be used to identify areas of the heartthat are ischemic but still viable as well as area that have lost theirviability due to infarction. These maps are based onelectrophysiological data that indicate the flow of activation signalsthrough the heart tissue. In addition, the data may be biomedical and/ormechanical data for example, variations in the thickness of the heartwall between systolic and diastolic stages of the heart cycle. The datathat is used to analyze the heart by mapping may also be a combinationof electrophysiological and biomedical data in order to more accuratelylocate and target the infarct region. In absence of viability mappingdevices, it is appreciated that the location of the infarction may bealso assessed through LV angiography or echo, where location of theakinetic or hypokinetic region may be identified.

Gene Manipulation

Genes that encode polypeptides harboring an antigenic determinantrecognized by the recipient organism or polypeptides associated with thesynthesis of molecules comprising an antigenic determinant recognized bythe recipient organism may be identified using a number of techniquesfamiliar to those skilled in the art. For example, in one embodiment,cDNA libraries are prepared from mRNA from the donor organism, or from aparticular cell type, organ or tissue from the donor organism that is tobe used in xenotransplantation. A variety of techniques are availablefor preparing cDNAs.

The resulting cDNAs are inserted into an expression vector such thatthey are operably linked to a promoter. Preferably, the expressionvector also encodes a marker that allows cells containing the expressionvector to be distinguished from cells that do not contain the expressionvector. For example, the marker may be a selectable marker that allowscells containing the vector to replicate in the presence of a drug.Alternatively, the marker may be a polypeptide which is easily detected,such as green fluorescent protein, red fluorescent protein, CD8, flagtag, HA tag, C-myc, GST, mbp, polyhistidine, and the like. Preferably,in order to facilitate identification of genes that encode polypeptidesfrom the donor organism that harbor an antigenic determinant, only asingle cDNA is introduced into each of the host cells. This may beachieved, for example, by infecting the host cells with retrovirusesencoding the polypeptide at a level of multiplicity such that each cellis only infected by a single virus.

The cells expressing the polypeptides encoded by the cDNAs from thedonor organism are contacted with naturally occurring immunoglobulinfamily proteins from the host subject. “Naturally occurringimmunoglobulin family proteins” may be defined broadly as polypeptidesthat contain an immunoglobulin domain, and occur naturally in theproposed recipient organism. These proteins, upon contact withpolypeptides, lipids, carbohydrates, and any other molecule including anantigenic determinant, may be capable of signaling the presence of anantigenic determinant that is recognized as associated with a recipientorganism. Those skilled in the art will appreciate that naturallyoccurring immunoglobulin family proteins include many different types ofmolecules and are present on the surface of many different cells. Forexample, without limitation, the naturally occurring immunoglobulinfamily proteins may be present in one or a combination of any of thefollowing: sera from one or more recipient organisms, such as humanbeings; a polyclonal antibody population or an enriched polyclonalantibody population from one or more recipient organism; any otherimmunoglobulin(s) from one or more recipient organism; or be present onthe surface B-cells, T-cells, including CD4+ and/or CD8+ cells,dendritic cells, macrophages and natural killer cells (NK cells) fromone or more recipient organism; and any other suitable cell or moleculefrom one or more recipient organism. Thus, the term “naturally occurringimmunoglobulin family proteins” include but are not limited toantibodies, B-cell receptors, T-cell receptors, MHC molecules, cellularreceptors, and cell surface molecules.

The expression vectors are introduced into host cells in which thepromoter is functional such that the polypeptides encoded by the cDNAsare produced in the host cells. The host cells may be any type of cellcapable of expressing the polypeptides encoded by the cDNAs from thepromoter. For example, the host cells may be mammalian cells. Themammalian host cells may be swine cells.

Another approach to identify antigenic determinants is to use eithercells from pig tissue or human cells, HeLa cells to HEK 293T cellsexpressing the cDNA library in the pCDNA3 vector. These cells can besubjected to subcellular fractionation to purify the cell surfacefraction. The proteins in the cell surface fraction may be subjected totwo-dimensional gel electrophoresis followed by SDS-PAGE. This gel isthen transferred to nitrocellulose and probed with the naturallyoccurring immunoglobulin family proteins derived from the recipient,which may or may not have been pre-absorbent on a column removing theantibodies directed against an unwanted surface molecule.

The genes encoding the polypeptides harboring an antigenic determinantrecognized by the recipient organism are sequenced using standardtechnology. To prevent or reduce recognition of the identifiedpolypeptides by the recipient organism, a desired number of the genesencoding the polypeptides are disrupted in cells from the donororganism. In one embodiment, only one gene is disrupted (e.g.,α-1,3-galactosyltransferase (GGTA1). In another embodiment, two, three,four, five, ten, twenty or more genes may be disrupted. The genes may bedisrupted in any cell from the donor organism that is capable of beingused to replace a cell or tissue in a xenotransplantation procedure. Forexample, the genes may be disrupted in cardiomyocytes, granulosa cells,muscle cells and primary fetal fibroblasts, stem cells (hematopoeticstem cells, bone marrow derived stem cells), germ cells, fibroblasts ornon-transformed cells from any desired organ or tissue. In oneembodiment, the genes may be disrupted in a stem cell population forregeneration of cardiomyocytes within a damaged region.

The genes may be disrupted using a variety of technologies familiar tothose skilled in the art. For example, a stop codon may be introducedinto the gene by homologous recombination. Alternatively, a deletion maybe introduced into the gene by homologous recombination. In someembodiments, stop codons may be introduced in all reading frames in thesequence downstream of the deletion to eliminate artifactual translationproducts. In further embodiments, the gene may be disrupted by insertinga gene encoding a marker protein therein via homologous recombination.It will be appreciated that the deletion, stop codon, marker gene, orother disruption may be located at any position which prevents orreduces recognition of the antigenic determinants by the immune systemof the recipient organism. If the donor cells are diploid, bothchromosomal copies of the gene may be disrupted in the donor cells.

Genes encoding polypeptides harboring antigenic determinants recognizedby the recipient organism may be sequentially disrupted in cells fromthe donor organism to generate cells in which each of the desired geneshas been disrupted. If desired, after disruption of each of the genes inthe cells from the donor organism, the cells may be contacted with serumfrom the recipient organism to confirm that recognition of thepolypeptides encoded by the genes by the recipient organism may beconsiderably reduced or eliminated. The disruption procedure may berepeated until cells from the donor organism having the desired numberof genes disrupted have been generated.

In some embodiments, the donor cells having the sought after disruptedgenes may be used to replenish cell populations (e.g., cardiomyocytes)or replace tissues. A variety of techniques may be used to generate thecell populations, or tissues. For example, in one embodiment, the donorcells may be used to generate a genetically modified organism, such as aknockout animal (e.g., a knockout swine or monkey) for example,including tissues or organs in which the desired genes have beendisrupted in most or all tissues and organs. A variety of techniques forgenerating transgenic or genetically modified animals are familiar tothose skilled in the art. For example, the nuclei of the donor cells maybe removed and transferred into enucleated oocytes capable of developinginto a transgenic or genetically modified animal. The oocytes may befrom the same species as the donor cells or from a different species.The oocytes including the nuclei from the donor cells may then beintroduced into an organism in which they can develop into a transgenicor genetically modified animal. The oocytes may be introduced into anorganism from the same species as the donor cells and/or the oocytes orfrom a different species from the donor cells and/or the oocytes. Theoocytes may be allowed to develop into genetically modified organismsand, after birth, the transgenic or genetically modified organisms areallowed to grow until their tissues or organs are suitable for use in axenotransplantation procedure. The genetically modified animal may alsobe generated by co-injection of the components necessary to induce thehomologous recombination together with sperm into the oocyte.

Alternatively, the donor cells with the desired genes disrupted may begenerated artificially by a simulated scaffold that forms the supportfor the tissue or organ. The scaffold may be a synthetic polymer or mayhave a biological component, such as a collagen. Such matrices have beendescribed in U.S. Pat. No. 6,051,071 (incorporated in its entirety).Donor cells having the desired genes disrupted may be grown on thescaffold. The scaffold comprising the donor cells is then implanted intothe recipient organism. On the other hand, the donor cells alone aredelivered to the target site such as the infarct zone.

In another embodiment, donor cells having the desired genes disruptedmay be genetically engineered to express a polypeptide beneficial to therecipient. For example, the donor cells may be genetically engineered toexpress a growth factor or cytokine as well as have a disrupted gene. Inanother embodiment, the vector may encode a factor that inhibits theactivity or reduces the amount of a nucleic acid, polypeptide,carbohydrate, lipid or any other molecules whose production may affectthe condition in a negative manner. Theses cells would also contain thedesired disrupted gene trait. In another embodiment molecules areexpressed by the transgenic or genetically modified animal that diminishrejection of the transplanted organ, tissue or cells in combination withthe disrupted gene phenotype.

Preparation of cDNA Libraries

cDNA libraries may be prepared from polyA+ RNA from the organism, celltype, tissue, or organ that is the donor in xenotransplantation. Forexample, if the donor organism is a pig, the mRNA may be prepared fromany desired cells, tissue or organ, including but not limited to kidney,liver, pancreas, heart, heart valve, lung, intestine, brain, cornea,endothelial cells or peripheral blood cells. If desired, the cDNAlibraries may be obtained from a commercial source such as Clontech(Palo Alto, Calif.) after supplying the source with tissue, total RNA orpolyA+ mRNA.

Alternatively, cDNA libraries are prepared using polyA+ RNA isolatedfrom donor organs obtained from a local slaughterhouse. In oneembodiment, the mRNAs may be obtained from one or more of the followingorgans including, but not limited to, the heart, heart valve, cornea,lung, intestine, or muscle and endothelial cells from large vessels.

An RNA preparation kit may be obtained from Invitrogen (Carlsbad,Calif.) The mRNA may be prepared according to the manufacturer'sinstructions or as known in the art. Briefly, the selected organs may beindividually homogenized and the cells may be lysed in RNase free lysisbuffer. The lysate may be passed through an 18-21 gauge needle. PolyA+RNA may be isolated by incubating the lysate with oligo(dt) cellulose inbatch and rotating. The oligo(dt) cellulose may be loaded onto a columnand extensively washed before the RNA is eluted off the oligo(dt)cellulose. The quality and the quantity of the mRNA may be monitored byvisualization of the mRNA by agarose gel electrophoresis and by opticaldensity (OD), respectively.

The mRNA obtained as described above may then used to prepare doublestranded cDNA using a modification of the protocol described in Huynh,et al., DNA Cloning, 1984, pp. 49-78, 1, the disclosure of which isincorporated herein by reference in its entirety. Briefly, mRNA isconverted into double-stranded DNA having unique ends that facilitatedirectional cloning into a vector, such as a retrovirus vector. First,the mRNA is hybridized to a linker-primer that incorporates a poly(dt)tract (at its 3′ end) as well as a restriction site for Not I. Thelinker-primer is extended using an RNase H⁻ version of the Moloneymurine leukemia virus transcriptase (Super Script, Gibco, BRL) and anucleotide mix in which dCTP is replaced with 5-methyl-dCTP. When firststrand synthesis is completed, the reaction mixture is transferred intoa second tube that contains the pre-chilled second-strand mixture. Thesecond strand is synthesized using RNAse H and E. Coli DNA polymerase I.Finally, a blunting step consisting of treatment with Mung bean nucleaseand Klenow fragment is carried out to prepare the cDNA for ligation to alinker, such as an EcoRI linker.

α-1,3-Galactosyltransferase (GGTA1) Knockout Cells

One approach for generating a transgenic animal that producesimmunotolerant cells involves micro-injection of naked DNA into a cell,preferentially into a pronucleus of an animal at an early embryonicstage (usually the zygote/one-cell stage). The production of oneimmunotolerant cell, the α-1,3-galactosyltransferase (GGTA1) knockoutswine cells, is incorporated here in its entirety (U.S. Pat. No.6,153,428). DNA injected as described integrates into the native geneticmaterial of the embryo, and will be replicated together with thechromosomal DNA of the host organism. Allowing the transgene to bepassed to all cells of the developing organism including the germ line.Transgene DNA that is transmitted to the germ line gives rise totransgenic offspring. All transgenic animals (50% of the offspring)derived from one founder animal are referred to as a transgenic line. Ifthe injected transgene DNA integrates into chromosomal DNA at a stagelater than the one cell embryo not all cells of the organism will betransgenic, and the animal is referred to as being genetically mosaic.Genetically mosaic animals can be either germ line transmitters ornon-transmitters. The general approach of microinjection of heterologousDNA constructs into early embryonic cells is usually restricted to thegeneration of dominant effects, i.e., one allele of the transgene(hemizygous) causes expression of a phenotype. See Palmiter, et al.,Ann. Rev. Genetics, 1986, p. 465, 20.

In a different approach, animals may be genetically altered by embryonicstem (ES) cell-mediated transgenesis (Gossler et al. 1986, Proc. Natl.Acad. Sci. USA. 83:9065). ES cell lines may be derived from earlyembryos, either from the inner cell mass (ICM) of a blastocyst (anembryo at a relatively early stage of development), or migratingprimordial germ cells (PGC) in the embryonic gonads. They have thepotential to be cultured in vitro over many passages (i.e., areconditionally immortalized), and they are pluripotent, or totipotent(i.e., are capable of differentiating and giving rise to all celltypes). ES cells can be introduced into a recipient blastocysttransferred to the uterus of a foster mother for development to term. Arecipient blastocyst injected with ES cells can develop into a chimericanimal, due to the contributions from the host embryo and the embryonicstem cells. ES cells can be transfected with heterologous geneconstructions that may cause either dominant effects, inactivate wholegenes or introduce subtle changes including point mutations. Subsequentto clonal selection for defined genetic changes, a small number of EScells can be reintroduced into recipient embryos (blastocysts ormorulae) where they potentially differentiate into all tissues of theanimal including the germ line and thus, give rise to stable lines ofanimals with designed genetic modifications. Totipotent porcineembryonic stem cells can be genetically altered to have a heterozygous(+/−) mutant, preferably null mutant allele, particularly one producedby homologous recombination in such embryonic stem cells. Alternatively,gene targeting events by homologous recombination can be carried out atthe same locus in two consecutive rounds yielding clones of cells thatresult in a homozygous (−/−) mutant, preferably a null mutant. SeeRamirez-Solis, et al., Methods in Enzymol, 1993, p. 855, 225.

In one preferred embodiment of this invention, a DNA sequence isintegrated into the native genetic material of the swine and producesantisense RNA that binds to and prevents the translation of the nativemRNA encoding. α-1,3-galactosyltransferase in the transgenic swine.

In a particularly preferred embodiment, the genome of the transgenicswine is modified to include a construct comprising a DNA complementaryportion of the α-1,3-galactosyltransferase coding region that willprevent expression of all or part of the biologically active enzyme.

In another embodiment of the invention, cells or cell lines fromnon-mutant swine are made with the α-1,3-galactosyltransferaseinactivated on one or both alleles through the use of an integratedantisense sequence which binds to and prevents the translation of thenative mRNA encoding the α-1,3-galactosyltransferase in the cells orcell lines. The integrated antisense sequence, such as the RNA sequencetranscribed, is delivered to the cells by various means such aselectroporation, retroviral transduction or lipofection.

In another preferred embodiment, the transgenic swine is made to producea ribozyme (catalytic RNA) that cleaves the α-1,3-galactosyltransferasemRNA with specificity. Ribozymes are specific domains of RNA which haveenzymatic activity, either acting as an enzyme on other RNA molecules oracting intramolecularly in reactions such as self-splicing orself-cleaving. See Long, D. M., et al., FASEB Journal, 1993, pp. 25-30,7.

The DNA for the ribozymes is integrated into the genetic material of ananimal, tissue or cell and is transcribed (constitutively or inducibly)to produce a ribozyme which is capable of selectively binding with andcleaving the α-1,3-galactosyltransferase mRNA.

In another preferred embodiment, using cultured porcine embryonic stemcells, a mutation, preferably a null mutation may be introduced by genetargeting at the native genomic locus encoding.α-1,3-galactosyltransferase. Gene targeting by homologous recombinationin ES cells is performed using constructs containing extensive sequencehomology to the native gene, but specific mutations at positions in thegene that are critical for generating a biologically active protein.Therefore, mutations can be located in regions important fortranslation, transcription or those coding for functional domains of theprotein. Selection for ES clones that have homologously recombined agene targeting construct, also termed gene “knockout” construct, may beachieved using specific marker genes. The standard procedure is to use acombination of two drug selectable markers including one for positiveselection (survival in the presence of drug, if marker is expressed) andone for negative selection (killing in the presence of the drug, ifmarker is expressed). See Mansour, et al., Nature, 1988, p. 348, 336.One preferred type of targeting vector includes the neomycinphosphotransferase (neo) gene for positive selection in the drug G418,as well as the Herpes Simplex Virus-thymidine kinase (HSV-tk) gene forselective killing in gancyclovir. Drug selection in G418 andgancyclovir, also termed positive negative selection (PNS), allows forenrichment of ES cell clones that have undergone gene targeting, ratherthan random integration events. See Mansour, et al., Nature, 1988, p.348, 336; Tubulewicz, et al., Cell, 1991, p. 1153, 65. Confirmation ofhomologous recombination events is performed using Southern analysis.

In another embodiment of the invention, cells or cell lines fromnon-mutant swine are made with the α-1,3-galactosyltransferaseinactivated on one or both alleles through the use of an integratedribozyme sequence which binds to and cleaves the native mRNA encodingthe α-1,3-galactosyltransferase in the cells or cell lines. Theintegrated ribozyme sequence, such as the RNA sequence transcribed isdelivered to the cells by various means such as electroporation,retroviral transduction or lipofection.

The swine may be preferably an α-1,3-galactosyltransferase negativeswine grown from a porcine oocyte whose pronuclear material has beenremoved and into which has been introduced a totipotent porcineembryonic stem cell using protocols for nuclear transfer. See Prather,et al., Biol. Reprod., 1989, p. 414, 41. ES cells used for nucleartransfer are negative for the expression of α-1,3-galactosyltransferase, or alternatively, totipotent ES cells used for nucleartransfer are mutated in a targeted fashion in at least one allele of theα-1,3-galactosyltransferase gene.

The swine is preferably lacking expression of theα-1,3-galactosyltransferase gene and bred from chimeric animals that aregenerated from ES cells by blastocyst injection or morula aggregation.ES cells used to generate the null-mutated chimeric animal may bemutated at least in one allele of the α-1,3-galactosyltransferase genelocus, using gene targeting by homologous recombination.

A chimeric swine is preferably constituted by ES cells mutated in oneallele of the α-1,3-galactosyltransferase gene. Derived from mutated EScells are also germ cells, male or female gametes that allow themutation to be passed to offspring, and allow for breeding ofheterozygous mutant sibling pigs to yield animals which are homozygousmutant at the α-1,3-galactosyltransferase locus. Also described is aswine, deficient for an α-1,3-galactosyltransferase protein (i.e.,characterized by lack of expression of α-1,3-galactosyltransferaseprotein) and have little, if any, functional Galα1-3Galβ1-4GlcNAcepitope-containing carbohydrate antigen on the cell surface areproduced. Further described are methods of producing transgenic swineand methods of producing tissue from heterozygous swine or homozygousswine of the present invention. The present invention also relates tocell lines, such as swine cell lines, in which theα-1,3-galactosyltransferase gene is inactivated on one or both allelesand use of such cell lines as a source of tissue and cells fortransplantation.

Tissues, organs and purified or substantially pure cells obtained-fromtransgenic swine, more specifically from hemizygous, heterozygous orhomozygous mutant animals may be used for xenogeneic transplantationinto other mammals including humans in which tissues, or cells may berequired. The α-1,3-galactosyltransferase inactive cells may themselvesbe the treatment or therapeutic/clinical product. In another embodiment,α-1,3-galactosyltransferase inactive cells produced by the abovedescribed method may be further manipulated, using known methods, tointroduce a gene or genes of interest, which encode(s) a product(s),such as a therapeutic product, to be provided to a subject. In thisembodiment, the α-1,3-galactosyltransferase deficient tissue, organ orcells serve as a delivery vehicle for the encoded product(s). Forexample, cytokines that augment donor tissue engraftment, Factor VIII,Factor IX, erythropoietin, insulin, human major histocompatibility (MHC)molecules or growth hormone, may be introduced.

A Knockout Swine Cell by Homologous Recombination

Gene targeting by homologous recombination in swine requires severalcomponents, including the following: (A) a mutant gene targetingconstruct including the positive/negative drug-selectable marker genes(see Tubulewicz, et al., Cell, 1991, p. 1153, 65); (B) embryonic stemcell cultures; and (C) the experimental embryology to reconstitute ananimal from the cultured cells.

The targeting construct may be provided from a genomic clone that spansmost of the antigenic determinant gene and is isolated from a librarymade of isogenic DNA from a major histocompatibility complex (MHC)haplotype d/d of the miniature swine. Fragments of that genomic cloneare introduced into a positive/negative selectable marker cassettespecifically developed for gene targeting in embryonic stem (ES) cellsand termed pPNT. See Tubulewicz, et al., Cell, 1991, p. 1153. 65. Thisgene targeting cassette may include as positive selectable marker thebacterial neomycin phosphotransferase gene (neo) which allows forselection of cells in G418. The neo gene is regulated by a promoter thatguarantees high level expression in ES cells such as thephosphoglycerate kinase promoter-1 (PGK-1). Negative selection isaccomplished by expressing the Herpes Simplex Virus-thymidine kinase(HSV-tk) gene that allows for selective killing of cells in Gancyclovir.Similar to the neo gene, the HSV-tk gene is regulated by the PGK-1promoter, as well. In the targeting cassette PPNT, there are unique andconvenient cloning sites between the neo and the HSV-tk gene which aresuitable sites to introduce the genomic fragment of the antigenicdeterminant gene upstream of the translation initiation signal AUG. Thisfragment of approximately 2 kb of DNA is cloned in reverse orientationto the direction of transcription of the PGK-neo cassette to assure thatno truncated or residual peptide is generated at the antigenicdeterminant-locus. Genomic sequences of the antigenic determinant locusdownstream may be introduced into PPNT at the 5′-end of the neo gene.This targeting construction termed pPNT-alpha GT1 is linearized andtransfected by electroporation into cells such as porcine ES cells.Double selection in G418 (150 to 300 μg/mL) and Gancyclovir is performedto initially isolate clones of ES cells with targeted mutations in thelocus. Confirmation of homologous recombinant clones is achieved usingSouthern analysis.

ES cell clones that have undergone targeted mutagenesis of one allele ofthe antigenic determinant (eg., α-1,3-galactosyltransferase) locus aresubjected to a second round of in vitro mutagenesis or used forreconstituting an animal that contains the mutation. A second round ofin vitro mutagenesis may be carried out using an analogous targetingconstruction with a positive selectable marker gene (eg., hygromycinphosphotransferase hyg).

As for reconstitution of animals, the methods include nuclear transfer,blastocyst injection or morula aggregation. The preferred routes includeeither blastocyst injection or morula aggregation which yield chimerasbetween the donor cells and the recipient embryos. For both thesemethods, recipient embryos are prepared as follows: embryodonor/recipient gilts are synchronized and mated. On day 6 followingartificial insemination or natural mating, the gilts may be prepared forsurgery as described earlier, anesthetized and the uteri retrogradelyflushed using a prewarmed (38.degree. C.) solution of phosphate bufferedsaline (PBS). Intact blastocysts that are encapsulated by the zonapellucida are placed in a depression slide containing HEPES-bufferedmedium (Whitten's or TL-HEPES) and approximately 15 to 20 ES cells areinjected. Injected embryos may be reimplanted into recipient fostergilts for development to term and pregnancies may be monitored usingultrasound. Offspring may be analyzed for chimerism using the polymerasechain reaction (PCR) of DNA samples extracted from blood, skin andtissue biopsies and primers complementary to the neo or hyg gene. Germline transmission of the chimeras is assayed using PCR and in situhybridization of tissue samples obtain from male and female gonads. Maleand female chimeras which transmit the ES cell genotype to the germ lineare crossed to yield homozygously mutant animals. Analysis of mutantanimals for expression of the antigenic determinant (e.g.,α-1,3-galactosyltransferase) and binding of human natural antibodies toendothelial cells of those animals is used as final test to assess thevalidity of gene knockout approach in swine.

Delivery Systems

Any one or more catheters may be used to deliver the any one or multiplecomponents of the embodiments to the infarct region area. Severalcatheters have been designed in order to precisely deliver agents to adamaged region within the heart for example an infarct region. Severalof these catheters have been described (U.S. Pat. Nos. 6,102,926,6,120,520, 6,251,104, 6,309,370; 6,432,119; 6,485,481). The deliverydevice may include an apparatus for intracardiac drug administration,including a sensor for positioning within the heart, a delivery deviceto administer the desired agent and amount at the site of the positionsensor. The apparatus may include, for example, a catheter body capableof traversing a blood vessel and a dilatable balloon assembly coupled tothe catheter body comprising a balloon having a proximal wall. A needlemay be disposed within the catheter body and includes a lumen havingdimensions suitable for a needle to be advanced there through. Theneedle body includes an end coupled to the proximal wall of the balloon.The apparatus also includes an imaging body disposed within the catheterbody and including a lumen having a dimension suitable for a portion ofan imaging device to be advanced there through. The apparatus mayfurther include a portion of an imaging device disposed within theimaging body adapted to generate imaging signal of the infarct regionwithin the ventricle. The apparatus may be suitable for accuratelyintroducing a treatment agent at a desired treatment site.

In another embodiment a needle catheter used to deliver the agent to theventricle for example, the infarct region, may be configured to includea feedback sensor for mapping the penetration depth and location of theneedle insertion. The use of a feedback sensor provides the advantage ofaccurately targeting the injection location. Depending on the type ofagent administered, the target location for delivering the agent mayvary. For example, one agent may require multiple small injectionswithin an infarct region where no two injections penetrate the samesite.

In other embodiments, the catheter assembly may include a maneuverableinstrument. This catheter assembly includes a flexible assembly. Thecatheter assembly may be deflectable and includes a first catheter, asecond catheter, and a third catheter. The second catheter fitscoaxially within the first catheter. At least one of the first catheterand the second catheter include a deflectable portion to allowdeflection of that catheter from a first position to a second position,and the other of the first catheter and second catheter includes aportion which is preshaped (e.g., an angled portion formed by twosegments of the angled portion). The third catheter has a sheath and amedical instrument positioned within the sheath. The third catheter fitscoaxially within the second catheter. In another embodiment, astabilizer, such as a donut shaped balloon, is coupled to a distalportion of the third catheter. Each catheter is free to movelongitudinally and radially relative to the other catheters. Thecatheter assembly may be used but not limited to the local delivery ofbioagents, such as cells used for cell therapy, one or more growthfactors for fibroblast retention, or vectors containing genes for genetherapy, to the left ventricle. In one embodiment, the catheter assemblydescribed may be used in delivering cell therapy for heart failure or totreat one or more portions of the heart that are ischemic. The catheterassembly uses coaxially telescoping catheters at least one or more beingdeflectable, to position a medical instrument at different targetlocations within a body organ such as the left ventricle. The catheterassembly may be flexible enough to bend according to the contours of thebody organ. The catheter assembly may be flexible in that the catheterassembly may achieve a set angle according to what the medical procedurerequires. The catheter assembly will not only allow some flexibility inangle changes, the catheter assembly moves in a three coordinate systemallowing an operator greater control over the catheter assembly'smovement portion of the second catheter, allowing for the distal tip ofthe third catheter to be selectively and controllably placed at amultitude of positions. It will be appreciated that the deflectableportion may alternatively be on the second catheter and the preshapedportion may be on the first catheter.

In a further embodiment, the apparatus of U.S. patent application Ser.No. 10/414,767 is incorporated here in its entirety. The apparatusincludes a first annular member having a first lumen disposed about alength of the first annular member, and a second annular member coupledto the first annular member having a second lumen disposed about alength of the second annular member, wherein collectively the firstannular member and the second annular member have a diameter suitablefor placement at a treatment site within a mammalian body.Representatively, distal ends of the first annular member and the secondannular member are positioned with respect to one another to allow acombining of treatment agents introduced through each of the firstannular member and the second annular member to allow a combining oftreatment agents at the treatment site. Such an apparatus isparticularly suitable for delivering a multi-component gel material(e.g., individual components through respective annular members thatforms a bioerodable gel within an infarct region of a ventricle).

In other embodiments, larger doses of treatment agent may be consideredfor example about 2 mL to about 250 mL that may require any one or moreof the delivery devices such as intra-venous retro infusion,intra-arterial infusion and needle catheter systems (INVIGOR availablefrom Abbott Vascular, Santa Clara, U.S.A.) as well as subxyphoidapproaches.

One concern of introducing any treatment agent composition, whetheradjacent to a blood vessel to affect therapeutic angiogenesis, adjacentto a tumor to inhibit tumor growth, or to induce or stimulate collagengrowth in arthroscopic procedures, is that the composition remains (atleast partially) at the treatment site for a desired treatment duration(or a portion of the treatment duration). In this manner, an accuratedosage may be placed at a treatment site with reduced concern that thetreatment agent will disperse, perhaps with serious consequences. In oneembodiment, a composition and technique for retaining a treatment agentat a treatment site (injection site) is described. In one embodiment, atreatment component (cellular component) and a bioerodable gel ornon-bioerodable gel or particle may be introduced at a treatment site(e.g., an injection site). The gel or particle(s) may be introducedprior to, after, or simultaneously with the treatment agent. In onepreferred embodiment, the gel or particle(s) acts to retain thetreatment agent at the treatment site by, representatively, sealing thetreatment site or sealing the treatment agent at the treatment site. Theuse of a gel or particle(s) with a treatment agent can reduce the amountof treatment agent backflow from the injection site as well as reducethe load requirement of the treatment agent at the treatment site. Forexample, a bioerodable product such as a gel or particle may decreasethe local pressure thereby further resulting in backflow reduction. Anon-bioerodable product may also decrease the local pressure to reducethe backflow in a more permanent fashion and at the same time may alsolead to an increase in compliance.

Using the above-mentioned techniques, an imaging modality or detectablemolecule may be added such as a contrast-assisted fluorescent scope thatpermits a cardiologist to observe the placement of the catheter tip orother instrument within the heart chamber. A contrast-assistedfluoroscopy utilizes a contrast agent that may be injected into heartchamber and then the area viewed under examination by a scope, thus thetopography of the region is more easily observed and may be more easilytreated (U.S. Pat. Nos. 6,385,476 and 6,368,285). Suitable imagingtechniques include, but are not limited to, ultrasonic imaging, opticalimaging, and magnetic resonance imaging, for example, Echo, ECG, SPECT,MRI, and Angiogram. Therefore, mapping of the heart is one techniquethat may be used in combination with the techniques proposed in thefollowing embodiments. In one embodiment, an echo angiograph may beperformed to confirm the occurrence and the location of the infarctregion. In another embodiment, a CAT scan may be performed to confirm anMI has occurred and the location of the infarct region. In anotherembodiment an EKG may be performed to identify the occurrence andlocation of an infarct. Another possibility is a molecule carried by acell such as a nucleic acid within a knockout cell (e.g., theα-1,3-galactosyltransferase (GGTA1) knockout cells) may be detectible bysequencing the molecule once the cells have been introduced to the site.

In another embodiment, a method may include introducing a treatmentagent in a sustained release composition. The preferred period forsustained release of one or more agents is for a period of one to twelveweeks, and in some cases two to eight weeks. Methods for local deliveryof sustained release agents include but are not limited to percutaneousdevices for example intraventricular (coronary) or intravascular(coronary and peripheral) devices.

Recruiting and Stimulating Agents

1. Agents

FIG. 4 describes one embodiment of a method to treat an infarct regionof a left ventricle. This is an illustrative diagram only and any of thetreatments may be used in parallel (e.g., at the same time) orsequentially or in any treatment combination. According to the methodillustrated in FIG. 4, a myocardial infarction may be detected by animaging process for example magnetic resonance imaging, optical imagingor ultrasonic imaging for example Echo ECG, spect, MRI, angiogram 410.Next, the area of the left ventricle is replenished by addition of acardiomyocyte replacement cell of an immunotolerant nature 420. Thengrowth factors may be introduced to stimulate the replacement cellpopulation to proliferate. In FIG. 4 one option to encourage thereplacement cell occupancy of the infarct zone includes the use ofscaffolding to generate a matrix for cellular adhesion 430 delivered tothe infarct zone. Suitable treatment agents that may modify thereplacement cell population include but are not limited to, AngiotensinII, fibroblast growth factor (FGF basic and acidic), insulin growthfactor (IGF), TGF-β in any of its isoforms (TGF-β 1, TGF-β 2, TGF-β 3),vascular endothelial growth factor (VEGF) in any of its isoforms, tumornecrosis factor-alpha (TGF-α), platelet-derived growth factor-BB(PDGF-BB), platelet-derived growth factor-AB (PDGF-AB), angiogenin,angiopoietin-1, Del-1, follistatin, granulocyte colony-stimulatingfactor (G-CSF), pleiotrophin (PTN), proliferin, transforming growthfactor-alpha (TGF-α), vascular permeability factor (VPF), stem cellfactor, stromal derived factor-1 (SDF-1), human growth factor (HGF) andLIH (leukemia inhibitory factor) genes that encode these proteins,transfected cells carrying the genes of these proteins, small moleculesand pro-proteins that also contain these proliferatory properties. Inone embodiment, growth factors such as bFGF, and/or VEGF may be used tomodify the region surrounding and/or within the infarct.

In one embodiment, a growth factor may be introduced to the infarctregion at the same time as another treatment by at least one of themethods described. In one embodiment, any of the described agents may beintroduced in one or more doses in a volume of about 1 μL to 1 mL. Inanother embodiment, any of the described agents may be introduced in oneor more doses in a volume of about 1 μL to 300 μL. In anotherembodiment, any of the described agents may be introduced in one or moredoses in a volume of about 1 μL to 100 μL. In a preferred embodiment,any of the described agents may be introduced in one or more doses in avolume of about 1 μL to 50 μL.

In alternate embodiments, the treatment volume may be larger (e.g.,intravenous pressure perfusion (IV) route). These volumes may range fromabout 2 mL to about 250 mL. Alternatively, these volumes may range fromabout 2 mL to about 100 mL. In other embodiments, these volumes mayrange from about 2 mL to about 30 mL.

2. Sequence of Treatment

FIG. 4 illustrates a flow chart of a process for treating MI byintroducing immunotolerant cells to the infarct region. FIG. 5A-5Eillustrates the introduction, action and proliferation of theimmunotolerant cells (e.g., α-1,3-galactosyltransferase (GGTA1) knockoutcells). Detection of acute myocardial necrosis may be performed using anECG (electrocardiogram) or by a more modern technology. For example, onetechnology such as ^(99m)Technetium-pyrophosphate or ¹¹¹In-antimyosinantibody imaging has recently been approved by the Food and DrugAdministration. With both of these two tracers, results are obtainedonly 24-48 hours after acute infarction and therefore, the clinicalutility of these techniques have been limited. There is another newagent called ^(99m)Tc-glucurate that produces results within an hourafter acute myocardial infarction (Iskandrian, A. S., et al., NuclearCardiac Imaging: Principles and Applications, Philadelphia, 1996). Oncethe MI is detected the exact location of the infarct may be identifiedusing a magnetic resonance imaging then the infarct region may betreated by reinforcement 510. An agent 520 (for example, immunotolerantcells) may be introduced to the infarct region 510. One way the agentmay be introduced to the area is percutaneously, with the use of acatheter. A distal end of the catheter is advanced to the infarct zone530, 540, or 550 and the agent 520 is released. Then for exampleimmunotolerant cells 560 remain in the site and/or proliferate andexpand 570. FIG. 5E illustrates the immunotolerant cell replacement andreinforcement of the infarct area.

In one embodiment, any of the described agents may be introduced in oneor more doses in a volume of about 1 μL to 1 mL. In another embodiment,any of the described agents may be introduced in one or more doses in avolume of about 1 μL to 300 μL. In another embodiment, any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 100 μL. In a preferred embodiment, any of the describedagents may be introduced in one or more doses in a volume of about 1 μLto about 50 μL.

Microparticles

One embodiment of a composition suitable for the described methodincludes the use of a bioerodable microparticle harboring one or morecomponent (eg. cells, growth factors) in combination withelectro-stimulus (eg. a pulse generator). The bioerodable microparticlemay consist of a bioerodable polymer such as poly(lactide-co-glycolide). The composition of the bioerodable polymer iscontrolled to release the component over a period of 1-2 weeks. It waspreviously demonstrated that biodegradable microparticles for example,poly (lactide-co-glycolide) were capable of controlled release of anoligonucleotide. These microparticles were prepared by the multipleemulsion-solvent evaporation technique. In order to increase the uptakeof the component into the microparticles it may be accompanied bypolyethylenimine (PEI). The PEI tends to make the microparticles moreporous thus facilitating the delivery of the oligonucleotide out of theparticles. See De Rosa, et al., Biodegradable Microparticles for theControlled Delivery of Oligonucleotides, Int. J. Pharm., Aug. 21, 2002,pp. 225-228, 242(1-2). In one preferred embodiment of a composition, abioerodable microparticle may be a PLGA polymer 50:50 with carboxylicacid end groups. PLGA is a base polymer often used for controlledrelease of drugs and medical implant materials (i.e., anti-cancer drugssuch as anti-prostate cancer agents). Two common delivery forms forcontrolled release include a microcapsule and a microparticle (e.g., amicrosphere). The polymer and the agent are combined and usually heatedto form the microparticle prior to delivery to the site of interest(Mitsui Chemicals, Inc). As the microparticles 580 erode 590, a porousnetwork of the microparticle composition is formed 595 in the infarctregion resulting in a matrix with a controlled pore size 595. As theporous network is formed in one example one angiogenic and/orpro-fibroblastic factor may be released encouraging the in-growth of newcapillaries. In one embodiment, the bioerodable polymer harbors acomponent such as the growth factor TGF-β1. In one embodiment, the PLGApolymer 50:50 with carboxylic acid end groups harbors TGF-β1 for slowrelease. It is preferred that each microparticle may release at least 20percent of its contents and more preferably around 90 percent of itscontents. In one embodiment, the microparticle harboring at least oneangiogenic and/or pro-fibroblastic agent will degrade slowly over timereleasing the factor or release the factor immediately upon contact withthe infarct area in order to rapidly recruit fibroblasts to the site. Inanother embodiment, the microparticles may be a combination ofcontrolled-release microparticles and immediate release microparticles.A preferred rate of deposition of the delivered factor will varydepending on the condition of the subject undergoing treatment.

Another embodiment of a composition suitable for the described methodincludes the use of microparticles that may harbor one or more of theaforementioned growth factors. The growth factors may be released fromthe microparticle by controlled-release or rapid release. Themicroparticles may be placed directly in the infarct region. By directlyplacing the particles in the infarct region, they may also provide bulkfor the region for reinforcement. The non-bioerodable microparticle mayconsist of a non-bioerodable polymer such as an acrylic basedmicrosphere for example a tris acryl microsphere (provided by BiosphereMedical). In one embodiment, non-bioerodable microparticles may be usedalone or in combination with an agent to increase compliance of aventricle. In another embodiment, non-bioerodable microparticles may beused alone or in combination with an agent to recruit fibroblasts and/orstimulate fibroblast proliferation. In addition, non-bioerodablemicroparticles may be used to increase compliance and recruitfibroblasts to an infarct region of a ventricle.

In one embodiment, the treatment agent compositions suitable forreinforcement of the infarct zone are rendered resistant to phagocytosisby inhibiting opsonin protein absorption to the composition of theparticles. In this regard, treatment agent compositions includingsustained release carriers include particles having an average diameterup to about 10 microns. In other situations, the particle size may rangefrom about 1 mm to about 200 mm. The larger size particles may beconsidered in certain cases to avoid macrophage frustration and to avoidchronic inflammation in the treatment site. When needed, the particlesize of up to 200 mm may be considered and may be introduced via anintraventricular catheter or retrograde venous catheter for any of theembodiments herein to avoid chronic inflammation due to macrophageinflux into the treatment site.

One method of inhibiting opsonization and subsequent rapid phagocytosisof treatment agents is to form a composition comprising a treatmentagent disposed with a carrier for example a sustained release carrierand to coat the carrier with an opsonin inhibitor. One suitableopsonin-inhibitor includes polyethylene glycol (PEG) that creates abrush-like steric barrier to opsonization. PEG may alternatively beblended into the polymer constituting the carrier, or incorporated intothe molecular architecture of the polymer constituting the carrier, as acopolymer, to render the carrier resistant to phagocytosis. Examples ofpreparing the opsonin-inhibited microparticles include the following.

For the encapsulation polymers, a blend of a polyalkylene glycol such aspolyethylene glycol (PEG), polypropylene 1,2-glycol or polypropylene1,3-glycol is co-dissolved with an encapsulating polymer in a commonorganic solvent during the carrier forming process. The percentage ofPEG in the PEG/encapsulating polymer blend is between five percent and60 percent by weight. Other hydrophilic polymers such as polyvinylpyrolidone, polyvinyl alchohol, or polyoxyethylene-polyoxypropylenecopolymers can be used in place of polyalkylene glycols, althoughpolyalkylene glycols and more specifically, polyethylene glycol isgenerally preferred.

Alternatively, a diblock or triblock copolymer of an encapsulatingpolymer such as poly (L-lactide), poly (D,L-lactide), or poly(lactide-co-glycolide) with a polyalkylene glycol may be prepared.Diblocks can be prepared by: (i) reacting the encapsulating polymer witha monomethoxy polyakylene glycol such as PEG with one protected hydroxylgroup and one group capable of reacting with the encapsulating polymer,(ii) by polymerizing the encapsulating polymer onto the monomethoxypolyalkylene glycol, such as PEG, with one protected group and one groupcapable of reacting with the encapsulating polymer; or (iii) by reactingthe encapsulating polymer with a polyalkylene glycol such as PEG withamino functional termination. Triblocks can be prepared as describedabove using branched polyalkylene glycols with protection of groups thatare not to react. Opsonization resistant carriers(microparticles/nanoparticles) can also be prepared using the techniquesdescribed above to form sustained-release carriers(microparticles/nanoparticles) with these copolymers.

A second way to inhibit opsonization is the biomimetic approach. Forexample, the external region of cell membrane, known as the“glycocalyx,” is dominated by glycoslylated molecules that preventnon-specific adhesion of other molecules and cells. Surfactant polymersmay consist of a flexible poly (vinyl amine) backbone randomlydistributed dextran and alkanoyl (hexanoyl or lauroyl) side chains whichconstrain the polymer backbone and lie parallel to the substrate.Hydrated dextran side chains protrude into the aqueous phase, creating aglycocalyx-like monolayer coating that suppresses plasma proteindeposition on the foreign body surface. To mimic glycocalyx,glycocalyx-like molecules can be coated on the carriers (e.g.,nanoparticles or microparticles) or blended into a polymer constitutingthe carrier to render the treatment agent resistant to phagocytosis. Analternate biomimetic approach is to coat the carrier with, or blend in,phosphorylcholine or a synthetic mimetic of phosphatidylcholine, intothe polymer constituting the carrier.

For catheter delivery, a carrier comprising a treatment agent (e.g., thecomposition in the form of a nanoparticle or microparticle) may besuspended in a fluid for delivery through the needle, at a concentrationof about one percent to about 20 percent weight by volume. In oneembodiment, the loading of the treatment agent in a carrier is about 0.5percent to about 30 percent by weight of the composition.Co-encapsulated with protein or small molecule treatment agents could bestabilizers that prolong the biological half-life of the treatment agentin the carrier upon injection into tissue. Stabilizers may also be addedto impart stability to the treatment agent during encapsulation.Hydrophilic polymers such as PEG or biomimetic brush-like dextranstructures or phosphorylcholine are either coated on the surface or thecarrier, grafted on the surface of the carrier, blended into the polymerconstituting the carrier, or incorporated into the moleculararchitecture of the polymer constituting the carrier, so the carrier isresistant to phagocytosis upon injection into the target tissuelocation.

Any one or more catheters may be used to deliver the any one or multiplecomponents of the embodiments to the infarct region area. Severalcatheters have been designed in order to precisely deliver agents to adamaged region within the heart, for example, an infarct region. Severalof these catheters have been described (U.S. Pat. Nos. 6,309,370;6,432,119; 6,485,481). The delivery device may include an apparatus forintracardiac drug administration, including a sensor for positioningwithin the heart, a delivery device to administer the desired agent andamount at the site of the position sensor.

Angiogenesis

After an MI, the infarct tissue as well as the border zone and theremote zone begin to remodel. The scar tissue forms in the infarctregion as the granulation is replaced with collagen, causing the scar tothin out and stretch. The perfusion in this region is typically 10% ofthe healthy zone, decreasing the number of active capillaries.Increasing the number of capillaries may lead to an increase incompliance of the ventricle due to filling up with blood. Other benefitsof increasing blood flow to the infarcted region include providing aroute for circulating stem cells to seed and proliferate in the infarctregion. Angiogenesis may also lead to increased oxygenation for thesurviving cellular islets within the infarct region, or to prime theinfarct region for subsequent cell transplantation for myocardialregeneration. In the border zone, surviving cells would also benefitfrom an increase in blood supply through an angiogenesis process. In theremote zone, where cardiac cells tend to hypertrophy and becomesurrounded with some interstitial fibrosis, the ability of cells toreceive oxygen and therefore function to full capacity are alsocompromised; thus, angiogenesis would be beneficial in these regions aswell. In one embodiment, angiogenesis will be stimulated in any regionof the heart—infarct, border or remote-through delivery ofangiogenesis-stimulating factors. Examples of these factors include butare not limited to isoforms of VEGF (e.g., VEGF121), FGF (e.g., b-FGF),Del 1, HIF 1-alpha (hypoxia inducing factor), PR39, MCP-1 (monocytechemotractant protein), Nicotine, PDGF (platelet derived growth factor),IGF (Insulin Growth Factor), TGF alpha (Transforming Growth Factor), HGF(Hepatocyte growth factor), estrogens, Follistatin, Proliferin,Prostaglandin E1, E2, TNF-alpha (tumor necrosis factor), Il-8(Interleukin 8), Hemotopoietic growth factors, erythropoietin, G-CSF(granulocyte colony-stimulating factors), PD-ECGF (platelet-derivedendothelial growth factor), Angogenin. In other embodiments, thesefactors may be provided in a sustained release formulation asindependent factor or in combination with other factors or appropriategene vectors with any of the gel or microparticle components describedin this application.

Microparticles and Angiogenic and Pro-Fibroblastic Agents

The microparticles may be prepared as microparticles harboring anangiogenic and/or pro-fibroblastic agent. On the other hand, themicroparticles may be prepared and then the angiogenic and/orpro-fibroblastic agent introduced into the microparticle, for example,by diffusion prior to introduction to the infarct region. In the laterexample, the microparticles might also be coated with the factor andupon introduction to the infarct region the factor immediately recruitsfibroblasts to the area. Additionally, the microparticle-factorcomposition might consist of any combination of the above-mentionedtreatments. In other embodiments, it may be necessary to add at leastone pharmaceutically acceptable inhibitor to the microparticles thatprevents decomposition of the angiogenic or pro-fibroblastic agent.

Microparticle Components

FIG. 4 describes a method to structurally reinforce the infarct regionby replacing damaged tissue. This method may be combined with any of themethods describing introducing angiogenic and/or fibroblast-recruitingagents, for example growth factors, to the infarct region to retainand/or promote fibroblast migration to this zone. This method may becombined with any of the methods describing introducing electricalstimulation to the infarct zone. Microparticles capable of taking upfluid will be introduced to the infarct region. Examples of thesemicroparticles include swellable non-biological or synthetic biologicalparticles. The microparticles may be introduced to the infarct zone andbecome trapped in the tissue. The microparticles tend to immediatelystart to swell. The swollen microparticles remain lodged in the tissueand provide reinforcement to the ventricular wall and add thickness tothe thinning infarct region.

The dimensions of the infarct zone may determine the size range of themicroparticles and the number of microparticles introduced to theinfarct region. This will insure that the optimum post-hydratedmicroparticle mass is achieved. An embodiment relates to microparticlesthat are about 200 microns or less in diameter. In another embodimentthe microparticles may be about 20 microns or less in diameter. In apreferred embodiment, the particle size may be about 5-10 microns indiameter. Particles of about 20 microns or less may also include anopsonization inhibitor (previously discussed). The swellablemicroparticles may be a range of sizes introduced to the infarct region.In one embodiment, the swellable non-biological material may be ahydrogel microsphere material. These microparticles are availablecommercially (A.P. Pharma or BioSphere Medical). These microparticlesare resistant to non-specific absorption and are bio-stable.Microparticles formed from the polymerization of an acidic monomer suchas Methacrylic acid may also be used. Microspheres containing carboxylicacid groups have been shown to be angiogenic in impaired wound healingmodels.

In other embodiments, the delivery of a nonbiologic or synthetic gel maybe combined with angiogenic and/or fibroblast recruiting agentsutilizing microparticles capable of releasing the agents at a rateoptimal for fibroblast retention and migration in the infarct region.

In one embodiment, immunotolerant cells suspended in a solution, such asa medium, may be introduced to the infarct region for structuralreinforcement of the ventricular wall. Media that may be used to supportthe growth and/or viability of the cells are known in the art andinclude mammalian cell culture media, such as those produced by GibcoBRL (Gaithersburg, Md.). See 1994 Gibco BRL Catalogue & Reference Guide.The medium can be serum-free but is preferably supplemented with animalserum such as fetal calf serum. Optionally, growth factors can beincluded. Media that are used to promote proliferation of cells andmedia that are used for maintenance of cells prior to transplantationcan differ. A preferred growth medium for cells such as a muscle cellmay be MCDB 120+dexamethasone, e.g., 0.39 μg/mL, +Epidermal GrowthFactor (EGF), e.g., 10 ng/mL, +fetal calf serum, e.g., 15%. A preferredmedium for muscle cell maintenance is DMEM supplemented with protein,e.g., 10% horse serum. Other exemplary media are taught, for example, inHenry, et al., Diabetes, 1995, p. 936, 44; WO 98/54301; and Li, et al.,Can. J. Cardiol., 1998, p. 735, 14.

Cells may be suspended in a solution or embedded in a support matrixwhen contained in an appropriate delivery device. As used herein, theterm “solution” includes a pharmaceutically acceptable carrier ordiluent in which the cells of the invention are suspended such that theyremain viable. Pharmaceutically acceptable carriers and diluents includesaline, aqueous buffer solutions, solvents and/or dispersion media. Theuse of such carriers and diluents is well known in the art. A solutionmay preferably be sterile and fluid. Preferably, the solution may bestable under the conditions of manufacture and storage and preservedagainst the contaminating action of microorganisms such as bacteria andfungi through the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention maybe prepared by incorporating cells as described herein in apharmaceutically acceptable carrier or diluent and, as required, otheringredients enumerated above, followed by filtered sterilization.

According to certain embodiments of the invention the composition maycontain compounds such as pharmaceuticals (e.g., antibiotic or agentsthat act on the heart), factors such as growth factors that maystimulate myoblast survival, proliferation, or differentiation, orfactors that may promote angiogenesis.

In one embodiment, delivery of cells or other components directly to thedamaged area of the heart may be accomplished with a catheter thataccesses the ischemic area of the heart and enters the myocardialtissue. For example, a catheter may be introduced percutaneously androuted through the vascular system or by catheters that reach the heartthrough surgical incisions such as a limited thoracotomy involving anincision between the ribs. The cells remain in the infarct region tofortify the tissue and enhance the modulus (wallstrength/elongation=modulus) of elasticity as well as replace the lostcardiomyocytes.

Methods for Introduction and Action

FIG. 5A-5E illustrates the introduction and action of cellularcomponents to the infarct region to replace dead cardiomyocyte cells.The cellular component may be introduced to the site 500 by a minimallyinvasive procedure 510. The solution may be injected in the infarct zoneduring an open chest procedure 520. The introduction of thepro-fibroblastic agent(s) includes one of the following procedures:sub-xiphoid and percutaneously 530. The mode of introduction of thepro-fibroblastic agent(s) by a percutaneous injection includes one ofthe following consisting of an intraventricular (coronary) catheter, atransvascular needle catheter, IC infusion and retrograde venousperfusion. One percutaneous route for a catheter is via a femoral arterytraversing through and then across the aortic arch into the leftventricle. Imaging techniques can guide the catheter to the infarctregion. The infarct region for example may be distinguished from healthytissue using MRI techniques. A catheter having imported the MRI data maythen be guided directly to the infarct region. The cellular component inthis aspect of the present invention may act as a structurallyreinforcing agent in the infarct zone 570. These cells add bulk to thearea and replace the degraded myocytes that without replacement maynormally lead to a thinning of the infarct regional wall. In turn, theviable cells release factors that may recruit other cells into the areafor further reinforcement of the infarct zone.

In one embodiment, any of the described agents may be introduced in oneor more doses in a volume of about 1 μL to 1 mL. In another embodiment,any of the described agents may be introduced in one or more doses in avolume of about 1 μL to 300 μL. In another embodiment, any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to about 100 μL. In a preferred embodiment, the any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to about 50 μL. If an agent is introduced via an IV or an ICroute the volumes may range from about 1 mL to about 500 mL.

Any one or more catheters may be used to deliver the any one or multiplecomponents of the embodiments to the infarct region area. Severalcatheters have been designed in order to precisely deliver agents to adamaged region within the heart for example an infarct region. Severalof these catheters have been described (U.S. Pat. Nos. 6,309,370;6,432,119; 6,485,481). The delivery device may include an apparatus forintracardiac drug administration, including a sensor for positioningwithin the heart, a delivery device to administer the desired agent andamount at the site of the position sensor.

Multiple Component Systems for Infarct Reconstruction Component One

To prevent heart failure, it has been proposed that cardiomyocytes canbe directly introduced into the infarct region to restore cardiacfunction cells of various origins, including embryonic and adult stemcells. One problem with this has been rejection by the host due to arigorous immune response. In addition, the viability of tissueengineering for a myocardial infarct zone requires that oxygen andnutrient supplies are readily available, as well as a mode for removalof waste products from cell metabolism. The cells in these areas alsoneed a supporting structure for adherence. The bioerodable gel withangiogenic agents previously discussed provides this later supportingstructure. In the literature, it is known that the introduction ofscaffolding with a bore size of less than 10 microns leads to a tightlyfibrotic encapsulated scaffold with poor capillary in-growth. On theother hand as demonstrated, if the scaffolding pore diameter is around20 microns, cellular encapsulation of the scaffold system is wellperfused with capillary in-growth leading to fibrotic poor cellular richregion. One embodiment includes scaffolding that is introduced to theinfarct zone and acts as a mechanical reinforcement. The force isdistributed more evenly at the infarct region and ventricular remodelingis prevented.

In one embodiment, separate components are included to provide a networksuch as described above. One example is described in FIG. 6. Amulti-component composition includes the first component including thepreviously illustrated bioerodable matrix or scaffolding 630. In thisparticular composition, the matrix (first component) provides a porousscaffolding to enhance capillary in-growth. The microparticles of thefirst component may be approximately 20 microns. In another embodiment,the first component of the composition may be introduced in a minimallyinvasive procedure such as percutaneously. A distal end of the catheteris advanced to the infarct zone and the bioerodable microparticles arereleased. In a further embodiment, the first component of thecomposition may be introduced via an intra-ventricular needle device tothe infarct region. In a further embodiment, an intra-ventricular needledevice including introducing multiple injections to the infarct regionmay introduce the first component of the composition. The firstcomponent may serve in one aspect as a domain to promote cell adhesionand growth (e.g., α-1,3-galactosyltransferase (GGTA1) knockout cells).In addition, porosity may be controlled that leads to capillaryin-growth. The first component may be a bioerodable microparticle withgrowth factor and angiogenic potential. The factor or other agent mayrelease over a 1-2 week period. One embodiment may be that the firstcomponent includes PLGA 50:50 (previously described) with carboxylicacid end groups. An example of capillary in-growth to the domainprovided by the first component may be facilitated by the release ofangiogenic factors. One embodiment includes microparticles containingangiogenic factors that release rapidly after introduction to theinfarct region. This tends to result in a rapid angiogenic response.

Biomaterials have been employed to conduct and accelerate otherwisenaturally occurring phenomena, such as tissue regeneration in woundhealing in an otherwise healthy subject; to induce cellular responsesthat might not normally be present, such as healing in a diseasedsubject or the generation of a new vascular bed to receive a subsequentcell transplant; and to block natural phenomena, such as the immunerejection of cell transplants from other species or the transmission ofgrowth factor signals that stimulate scar formation in certainsituations.

Component Two

A second component 640 of a multi component composition according to themethod may be stimulating the heart using a device such as a pacemaker.A second component serves in one aspect to stimulate the cardiacfunction. On the other hand, it also serves to encourage unloading ofthe damaged area. It may be accomplished by introducing one or moreleads, for example, to the infarct region. The scaffold may serve as acamouflage from the immune system for introduction of the microparticlesto the infarct region. The electrical stimulation serves to unload thearea and prevent remodeling. In addition, a cellular component mayconsist of a population that has an altered surface antigen to preventimmune recognition of α-1,3-galactosyltransferase (GGTA1) knockoutcells. One embodiment includes the injection of both the growth factorcontaining microparticles and the cellular component (e.g.,α-1,3-galactosyltransferase (GGTA1) knockout cells) using a dual boreneedle. As the microparticles decompose, growth factors may be releasedpromoting the capillary formation within the matrix. In addition, cellsbegin to grow in the infarct area. These cells release proteases thatmay result in the decomposition of the scaffolding, ultimately creatingadditional area for cellular in-growth. In addition, cells secrete theirown extracellular matrix, the polymer degrades and the resulting tissuemay eventually become a completely natural environment. Thedecomposition products may be cleared from the area by the renal systemsince capillary re-growth may occur.

Any one or more catheters may be used to deliver the any one or multiplecomponents of the embodiments to the infarct region area including, butnot limited to, a dual bore needle catheter. Several catheters have beendesigned in order to precisely deliver agents to a damaged region withinthe heart, for example, an infarct region. Several of these cathetershave been described (U.S. Pat. Nos. 6,309,370; 6,432,119; 6,485,481).The delivery device may include an apparatus for intracardiac drugadministration, including a sensor for positioning within the heart, adelivery device to administer the desired agent and amount at the siteof the position sensor.

Multi Component System for Infarct Reconstruction and InfarctReoxygenation

The progression of heart failure after an MI is a result of theremodeling of the heart after infarct. In the remodeling processes theheart becomes thinner and the diameter increases in response to adecrease in heart output, in an effort to maintain a continual cardiacoutput. This process of thinning results in an increase in the radius ofthe heart and the stresses on the heart increase.

It has been shown that perfluorocarbon compounds have a high affinityfor gases, for example carbon dioxide and oxygen. The ability ofperfluorocarbons to transport oxygen is approximately eighteen timesgreater than blood plasma in a comparable volume of each component. Inaddition, it was shown that the half-life for oxygenation/deoxygenationis approximately three and one half times faster for many perfluorinatedcompounds as compared to hemoglobin. Thus, perfluoro compounds may beused in tissues to aid in the reoxygenation of an affected region suchas an infarct region. A few examples that demonstrate biocompatibilityin a subject are identified in Table 1.

TABLE 1 Compound Properties Trade Name Vapor O₂ solubility or CommonMolecular Pressure at 37° C. Name Chemical Name and Structure Weight(mmHg) (V %) F-44E 1,2-bis(perfluorobutyl)ethane 462 12.6 50F₉C₄—CH═OH═C₄F₉ F-66E or 1-perfluoropropane-2-erfluorohexyl)ethane 6642.3 41 F-i66E F₇C₃—CH═CH═C₆F₁₃ FDC Perfluorodecalin 462 12.5 45 C₁₀F₁₈

In addition, any of the above detailed embodiments may be combined withelectrical stimulation to unload the infarct region. Electricalstimulation may be used before 760, simultaneously 770 and/or after 780treatment of reoxygenating compounds to an infarct region.

FIG. 7 illustrates the multi-component system in a flowchart. Themyocardial infarction is located 720. Then, the components are deliveredto the region via a minimally invasive procedure by methods previouslydescribed and/or by catheter delivery. It was previously disclosed thatthe addition of a thiol functionality U.S. patent application Ser. No.10/414,767. FIG. 6 component 3 in the presence of an electron deficientdouble bond, such as an acyloyl functionality FIG. 6 component 2, canundergo a Michael addition reaction. Under basic conditions a thiolfunctionality becomes hypernucleophilic and rapidly (<10 seconds) formsa bond with the acryloyl functionality. As illustrated in FIG. 6, a gelmay be formed to prevent infarct expansion and/or bulking thuspreventing a remodeling of the heart that may lead to heart failure.This may be combined with a stimulatory component FIG. 6 640 in thisapplication to possibly unload the infarct region. FIG. 7 730illustrates the first component that includes a bioerodable gel and 740illustrates the gel accompanied by a perfluorinated compound as thesecond component to enhance oxygenation of the tissue. The gel is formedby a three-component system. The first component includes abiocompatible polymer as previously described with a multifunctionalspacer group 730. The second component 740 includes a difunctional ormultifunctional perfluorinated molecule 740. The third component 750includes a hetero-functional molecule with a reactive functionality onone side of the spacer group, and a cell binding peptide sequence, suchas the peptide sequences previously described, on the terminal end. Oneexample of a peptide sequence includes an RGD sequence. Thisthree-component system may be introduced to the infarct region bysimilar minimally invasive methods as described for the methods of FIG.6 that may be guided by mapping the heart prior to administration of anagent. An electrical stimulus such as pulse-generating leads may beplaced in or around the infarct region and may be used at any timeduring a gel reinforcement treatment to unload the region.

In one embodiment, any of the described agents may be introduced in oneor more doses in a volume of about 1 μL to 1 mL. In another embodiment,any of the described agents may be introduced in one or more doses in avolume of about 1 μL to 300 μL. In another embodiment, any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 100 μL. In a preferred embodiment, the any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 50 μL. IV, and IC routes may be required which wouldinvolve larger treatment volumes (for example about 2 mL to about 250mL).

An ester bond may be formed between a second component and a thirdcomponent of a multi-component composition of, for example, FIG. 6. Thisbond is necessary for delaying the degradation of the scaffolding andrelease of the active agents within the microparticles. This bond tendsto resist degradation for approximately 2 months.

Swellable Agent Systems for Reinforcement

One method for introduction of the microparticles is percutaneously withthe use of a catheter. A distal end of the catheter may be advanced tothe infarct region and the microparticles may be released. Themicroparticles become lodged in the infarct tissue. One embodimentincludes the use of microparticle beads capable of fluid uptake in theinfarct region to structurally reinforce the region. The particles willrange in size from approximately 5 to approximately 10 microns. Themicroparticles will be less than 10 microns so that the completelyswollen particle becomes lodged in the site but is not too large tobecome an obstruction in the area. In addition, the swollenmicroparticles provide mechanical strength and thickness to the damagedarea by replacing the dead and degraded myocardial cells.

1. Agents

a. Hydrogels Spheres

Examples include hydrogel spheres composed of cross-linkedpolyacrylamide or cross-linked PVP. The monomeric form of these productswill contain di-functional monomers such as di-vinyl benzene, ethyleneglycol dimethylacrylate or bis acrylamido acetic acid. These agents forma cross-linked network that is resistant to dissolution in aqueoussystems.

b. Commercial Products

Several commercial products are available that may be used such asmicroparticles obtained from A.P. Pharma or Biosphere Medical. Thesemicroparticles resist non-specific protein absorption and havebio-stable backbone linkages. These microparticles are not bioerodableor bioabsorbable.

Structural Reinforcement Compositions and Materials

Several possible methods to reinforce the ventricular wall of theinfarct region of an MI subject. Restraining the infarct zone bysuturing an epicardial polymer mesh was previously demonstrated. SeeKelley, S. T., et al., Restraining Infarct Expansion Preserves LeftVentricular Geometry and Function After Acute Anteroapical Infarction,Circ., 1999, pp. 135-142, 99. Due to the nature of this techniquesuturing the mesh directly into the tissue was necessary. This may causefurther damage. This procedure requires invasive surgery. In addition,the polymer mesh does not degrade over time and this may also be aproblem. By injecting a reinforcing agent directly into the affectedarea by minimally invasive procedures, this avoids the intrusivesuturing protocol. The solution may be injected in the infarct zoneduring an open chest procedure. In one embodiment, the introduction ofthe reinforcing solution comprises the following procedures consistingof sub-xiphoid and percutaneously. In another embodiment, the mode ofintroduction of the reinforcing solution by a percutaneous injectioncomprises one of the following consisting of an intraventricularcatheter, a transvascular needle catheter and retrograde venousperfusion.

2. Single Component Systems

One embodiment utilizes a single pseudoplastic or thixotropic materialcapable of forming a gel-like reinforcement to the infarct region wall.Several examples of these materials exist. In one embodiment, thestructural reinforcing agent includes one of the following: hyaluronicacid, bovine collagen, high-molecular weight ultra-pure polyacrylamideand polyvinyl pyrrolidone.

In one specific embodiment of the present invention, the singlecomponent for structural reinforcement comprises bovine collagendispersed with PMMA (polymethyl methylacrylate) beads. These beads maybe manufactured under the trade name of ARTECOLL (Rofil MedicalInternational, Breda, The Netherlands). PMMA is one of severalcross-linked or highly insoluble microparticles. PMMA was discovered inthe early 1900's and was used initially in dental prosthesis. Recently,it has been used in bone replacement of the jaw and hip. In addition, ithas been used for artificial eye lenses, pacemakers and dentures.ARTECOLL™ has principally been used in filling folds and wrinkles of theface, augmenting lips, adjusting an irregular nose.

Possibly one of the most important features of the insolublemicroparticles is the surface of the microparticles must be smooth toinduce collagen deposition. A rough surface promotes macrophage activitywhile discouraging collagen deposition. The methods incorporate the useof smooth surface particles. The components may act as a substrate forendogenous collagen deposition. As the reinforcing gel degrades, thehighly stable and smooth microparticles may be exposed to the fibroblastcell population occupying the site. This triggers the production ofcollagen to replace the decomposing gel. Therefore, the infarct zone maybe reinforced by the collagen replacement of the temporary gel. In oneembodiment, the dispersing material includes one of the following groupof microparticle materials consisting of PMMA (polymethylmethylacrylate), P(MMA-co BMA) (polymethyl methylacrylate-co butylmethylacrylate), carbon microparticles (Durasphere), polystyrene,cross-linked acrylic hydrogels and PLGA. In another embodiment, thecross-linked acrylic hydrogel may include the following HEMA(2-hydroxyethyl methacrylate), AA (acrylic acid), AMPS(acrylamido-methyl-propane sulfonate), acrylamide, N, N, di-methylacrylamide, diacetone acrylamide, styrene sulfonate, and di- ortri-functional monomers. The di or tri-functional monomers may be EGDMA(ethylene glycol dimethacrylate) and DVB (di-vinyl benzene). Inaddition, the use of highly crystalline (and hydrolysis resistant) PLGAmicroparticles may outlast the carrier gel and also provide a usefulsubstrate for collagen deposition.

Another single solution introduced to the infarct zone may be hyaluronicacid dissolved in sodium salt in water. This is a manufactured gel soldas a dermal augmentation gel (RESTYLANE™). Hyaluronic acid hydrogel hasalso been used in the past for control of delivery of therapeutic agentsin wound sites. See Luo, Y., et al., Cross-linked Hyaluronic AcidHydrogel Films: New Biomaterials for Drug Delivery, Journal ofControlled Release, 2000, pp. 169-184, 69. Other possible singleintroduced components include bovine collagen (ZYDERM™ or ZYPLAST™),another dermal augmentation gel developed by Collagen Corp. The highmolecular weight, ultrapure polyacrylamide in water may be FORMACRYL™ orBIOFORM™ other dermal augmentation gels. The bovine collagen may bedispersed by the PMMA product ARTECOLL™. ARTECOLL™ is best known for itssuccess as a biocompatible dermal augmentation gel for reconstruction.RESOPLAST™ (Rofil Medical International, Breda, The Netherlands) mayalso be used as a single component gel.

In another embodiment, a reactive single component includes a componentthat is temperature sensitive. One example of this type of component isa component that may be a liquid at room temperature and once exposed toa temperature approximately equal to body temperature the componentgels. A more specific component includes introducing block co-polymersof silk protein-like sub units and elastin-like sub units. An example ofthe block co-polymer synthetic protein may be ProLastin (PPTI, ProteinPolymer Technologies). These components gel due to non-covalentinteractions (hydrogen bonding and crystallization of silk-likesubunits) at elevated temperatures for example approximately equal tobody temperature. With these components, no lysine residues are present,so cross-linking due to endogenous lysyl oxidase does not occur. Theformation of the gel via a change in temperature may be adjusted usingadditives. These additives include but are not limited to sodiumchloride, Diglyme (Diethylene Glycol Dimethyl Ether; 2-MethoxyethylEther; Bis(2-Methoxy Ethyl Ether), and ethanol.

Many thermal reversible materials may be used for reinforcement of themyocardial tissue. Generally, thermal reversible components attemperatures of approximately 37 degrees Celsius and below are liquid orsoft gel. When the temperature shifts to 37 degrees Celcius or above,the thermal reversible components tend to harden. In one embodiment, thetemperature sensitive structurally reinforcing component may be Triblockpoly (lactide-co-glycolide)-polyethylene glycol copolymer. This iscommercially available (REGEL™ Macromed, Utah). In another embodiment,the temperature sensitive structurally reinforcing component may includethe following consisting of poly (N-isopropylacrylamide) and copolymersof polyacrylic acid and poly (N-isopropylacrylamide). Anothertemperature sensitive structurally reinforcing component commerciallyavailable is PLURONICS™ (aqueous solutions of PEO-PPO-PEO (poly(ethyleneoxide)-polypropylene oxide)-poly(ethylene oxide) tri-block copolymersBASF, N.J.). See Huang, K., et al., Synthesis and Characterization ofSelf-Assembling Block copolymers Containing Bioadhesive End Groups,Biomacromolecules, 2002, pp. 397-406, 3. Another embodiment includescombining two or more of the single components in order to structurallyreinforce the infarct region. For example, silk-elastin, collagen andLaminin may be used as a one-part system. The silk-elastin would likelyform in situ cross-links due to the silk blocks.

In another embodiment, a reactive single component includes a componentthat is pH sensitive. The component remains in a liquid state if it issufficiently protonated preventing gelation. In another embodiment, thecomponent is initially maintained at a low pH for example pH 3.0 andlater introduced to the treatment area which results in gelation of thecomponent due to the physiological pH of the environment. Severalpossible cationic agents may be but are not limited to one of thefollowing cationic agents that remain protonated at low pH, poly (allylamine), DEAE-Dextran, ethoxylated Poly(ethylenimine), and Poly(lysine).Other examples may one of, but are not limited to, the following anionicagents: dextran sulfate, carboxymethyl dextran, carboxymethylcellulose,polystyrene sulfanate and chrondroitin sulfate.

Additionally, any of these microparticle components may be accompaniedby one or more contrast agent and/or suitable agent(s) for treatment ofthe region. The contrast agent or treatment agent may be conjugated toor dissolved into the structural component prior to introduction to theinfarct area. The agents that may accompany the reinforcing component(s)may include but are not limited to angiogenic agents, ACE inhibitors,angiotensin receptor blockers, SRCA (sercoplasmic reticulum calciumpump) pump increasing agents, phospholamban inhibitors andanti-apoptotic drugs. These agents may be in the form of smallmolecules, peptides, proteins or gene products. The small molecules maybe optionally conjugated to a component of the solution, dispersed insolution, or dissolved in solution to improve the adhesion of thereinforcing components to the tissue. One embodiment is to conjugate apeptide with a conserved region that mediates adhesion processes. Aconserved region of a peptide may be a sequence of amino acids having aspecial function of identification that has been conserved in a proteinfamily over time. Another embodiment includes the use of a specificpeptide conjugate with a conserved RGD (arginine(R)-glycine(G)-asparticacid (D)) motif in the presence of the reinforcing component. In furtherembodiments, the RGD motif peptide may include the following: vonWillebrand factor, osteopontin, fibronectin, fibrinogen, vitronectin,laminin and collagen. One embodiment seeks to minimize thinning duringremodeling of the infarct region. Thus, bulking and reinforcing theinfarct region post-MI may preserve the geometry of the ventricle.

Any one or more catheters may be used to deliver the any one or multiplecomponents of the embodiments to the infarct region area. Severalcatheters have been designed in order to precisely deliver agents to adamaged region within the heart for example an infarct region. Severalof these catheters have been described (U.S. Pat. Nos. 6,309,370;6,432,119; 6,485,481). The delivery device may include an apparatus forintracardiac drug administration, including a sensor for positioningwithin the heart, a delivery device to administer the desired agent andamount at the site of the position sensor.

Dual Component Systems

Dual component systems for the formation of structurally reinforcinggels for application to the infarct region may be used. Initially, theinfarct region is identified by imaging methods previously discussed. Inone example, two components are combined at the infarct zone at aroundphysiological pH. Component one is a principally anionic solution andthe second component is principally a cationic solution at approximatelyphysiological pH. When the two components are mixed together at theinfarct zone, a gel forms rapidly and irreversibly. In one embodiment, adual component system may comprise poly (acrylic acid) as a firstcomponent and poly (allyl amine) as a second component. In anotherembodiment, a dual component system may comprise poly (acrylic acid) asa first component and poly (allyl amine) as a second component that maybe delivered by a catheter with dual injection lumens. Other dualcomponent systems to form a structurally reinforcing gel in the infarctregion may include elastin as a first component and lysyl oxidase as asecond component; sodium alginate as a first component and an aqueoussolution of calcium chloride as a second component, and tropoelastin andcollagen as a first component and cross-linker lysyl dehydrogenase as asecond component and laminin may be added to this combination later. Thecomposition of each component will depend on the mechanical property ofthe final cross-linked system. Other substances that can replace thelysyl dehydrogenase or complement its cross-linking ability might beused such as glutaraldehyde, and/or photoactivatable crosslinkers forexample blue dye used to cross-link. Additionally, these dual componentsystems may be combined with other individual system utilizingcommercial products such as AVITENE™ (Microfibrillar Collagen Hemostat),SUGICEL™, (absorbable haemostat, Johnson & Johnson), GELFOAM™, FLOSEAL™(Baxter, matrix hemostatic sealant with a granular physical structureand thrombin), FOCAL SEAL™ (Focal, Inc.) or FIBRIN SEAL™ (FS). FLOSEAL™is a gel constituting collagen derived particles and topical thrombincapable of being injected. It has been approved for uses includingvascular sealing. Several other possible cationic agents may be but arenot limited to one of the following cationic agents that remainprotonated at low pH, poly (allyl amine), DEAE-Dextran, ethoxylatedPoly(ethylenimine), and Poly(lysine). Other examples may be one of butare not limited to the following anionic agents: dextran sulfate,carboxymethyl dextran, carboxymethylcellulose, polystyrene sulfonate andchrondroitin sulfate. In a preferred embodiment, the first material maybe DEAE Dextran and the second material may be polystyrene sulfonate.

One dual component system may use DOPA (3,4-dihydroxyphenyl-L-alanine),a principle component responsible for mussel adhesive proteins, capableof forming a hydrogel in conducive conditions. Specifically, a componentknown as star block DOPA-block-PEG undergoes cross-linking in situforming the hydrogel after an oxidation process converts the DOPA toO-quinone. This process forms a stable in situ hydrogel. A specificembodiment may include the use of PEG triacrylate as a first componentand PEG thiol as a second component introduced to the infarct zone via adual lumen needle system discussed previously. A glue-like componentsystem may be employed. One embodiment may include the use of GRF gluethat is made up of gelatin, resorcinol and formaldehyde (GRF) as astructurally reinforcing agent introduced to the infarct zone. Toaccomplish this, a two-part system may be used to induce cross-linkingupon admixture of the components at the infarct zone. In otherembodiments, the following structurally reinforcing components may beadded along with GRF comprising the group consisting of thecross-linking agents polyglutamic acid, polylysine and WSC (watersoluble carbodimides).

A single pseudoplastic or thixotropic agent may be introduced to aninfarct region in multiple injections and to structurally reinforce thewall. These agents are introduced in final form and require noadditional agents. Multiple injections each at a different site thatrequires an endogenous component or a temperature change to convert to astructurally reinforcing form may be used. The structurally reinforcingagent(s) is localized to the infarct region via minimally invasiveprocedures discussed previously.

In addition, biocompatible viscosifiers for example type 1 gels may beadded in combination with any of the single or multiple componentsystems illustrated. In addition electrical stimuli may be applied atany time of treatment to unload the infarct region. In one example,hyaluronic acid or PVP may be used to increase the resistance of theactive formula from natural degradation once introduced to the infarctzone. In one embodiment the viscosity of the treatment agent may beabout 0-100 centipoise. In other embodiments, the viscosity of thetreatment agent may be about 0-50 centipoise. In a preferred embodiment,the viscosity of the treatment agent may be about 25-40 centipoise. In apreferred embodiment, the viscosity of the treatment agent may be about35 centipoise.

In one embodiment, any of the described agents may be introduced in oneor more doses in a volume of about 1 μL to 1 mL. In another embodiment,any of the described agents may be introduced in one or more doses in avolume of about 1 μL to 300 μL. In another embodiment, any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 100 μL. In a preferred embodiment, the any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 50 μL. IV, and IC routes may be required which wouldinvolve larger treatment volumes (for example about 2 mL to about 250mL).

Biocompatible dyes may be added to any single or combination componentsof any of the described embodiments to trace the components in theinfarct region in any procedure. Other dyes may be added forexperimental purposes to trace the deposition of any agent for examplein a rat heart. Some examples of these dyes include but are not limitedto Sudan Red B, Fat Brown RR, Eosin Y and Toluidine blue.

On the other hand, tissue adhesive components may also be added incombination with any of the single or dual component systems. Forexample, Laminin-5, polyacrylic acid, Chitosan and water solublechitosan may be used to increase the tissue retention of the activeformulation. Laminin-5 is a basement membrane extracellular matrixmacromolecule that provides an attachment substrate for both adhesionand migration in a wide variety of cell types, including epithelialcells, fibroblasts, neurons and leukocytes. Chitosan is the only naturalpositive ion polysaccharide obtained from deacetylated chitin. Itpossesses decomposability, good membrane forming state,biocompatibility, anti-fungal and anti-tumor function. Chitosan hasexcellent viscosity, compressibility and fluidity.

Single Components Suspended in a Delivery Medium

As with several of the previously discussed methods, other methodsprovide a bulking or structurally reinforcing agent to the infarctregion. An agent comprising microparticles in solution (a dispersion)may be introduced to the infarct region after identification of theinfarct region as described previously. The microparticles may be apredetermined range of about 1 to about 200 microns. In one embodiment,the microparticles may be 20 microns or less. In a preferred embodiment,the microparticles may be 10 microns or less. The microparticle sizedelivered to an infarct region may be determined by the delivery methodused. For example an intraventricular catheter may be used to deliverparticles up to 200 microns that may avoid the risk of an embolism. Onesuspending solution for the microparticles may be water. On the otherhand, the suspending solution may also be a solvent, for example,dimethylsulfoxide (DMSO) or ethanol adjuvants. In one embodiment, asuspending solution along with the microparticles may be introduced toas a dispersion to an infarct region and the microparticles remain inthe region as the solution dissipates into the surrounding tissue. Thus,the microparticles provide a structurally reinforcing bulk to theregion. This may result in reduction of stress to the post infarctmyocardium. It may also serve as a substrate for additional site forcollagen deposition. In one embodiment, the dispersion (detailed above)may be injected in to the infarct zone during an open chest procedurevia a minimally invasive procedure. In another embodiment, the minimallyinvasive procedure includes at least one of subxiphoid andpercutaneously. In another embodiment, the percutaneous introductioninto the infarct zone may include one of intra-ventricular needle,transvascular catheter and retrograde venous perfusion.

Several examples of gels that may be used in any embodiment herein existsuch as the viscous liquid sucrose acetate isobutyrate (SAIB). SAIB iswater insoluble. SAIB may be dissolved in a solvent or a combination ofsolvents, for example, ethanol, dimethylsulfoxide, ethyl lactate, ethylacetate, benzyl alcohol, triacetin, 2-pyrrolidone, N-methyl pyrrolidone,propylene carbonate or glycofurol. These solvents decrease the viscosityof SAIB in order to facilitate the introduction of this agent through aneedle or lumen. In one embodiment, AIB may be introduced accompanied bya solvent to the infarct region and the solvent dissipates at the siteleaving behind the viscous SAIB in the region. In another embodiment, aSAIB treated infarct region may be accompanied by an electrical stimulussuch as a pulse generator to unload the area before, during or aftertreatment with SAIB.

Other biocompatible polymer systems may be introduced to an infarctzone. Some of these agents are not only biocompatible but alsosubstantially water insoluble similar to SAIB. Solvents or mixtures ofsolvents may be used to dissolve the polymer in order to facilitateintroduction to the infarct zone. In one embodiment, a biocompatiblewater insoluble polymer may include the following consisting ofpolylactides, polyglycolides, polycaprolactones, polyanhydrides,polyalkylene oxates, polyamides, polyurethanes, polyesteramides,polydioxanones, polyhydroxyvalerates, polyacetals, polyketals,polycarbonates, polyorthoesters, polyphosphazenes, polyhydroxybutyrates,polyalkylene succinates, and poly(amino acids). Any one of theseinsoluble polymers may be dissolved in solvents for example Diglyme,dimethyl isosorbide, N-methyl-2-pyrrolidone, 2-pyrrolidone, glycerol,propylene glycol, ethanol, tetraglycol, diethyl succinate, solketal,ethyl acetate, ethyl lactate, ethyl butyrate, dibutyl malonate, tributylcitrate, tri-n-hexyl acetylcitrate, dietyl glutarate, diethyl malonate,triethyl citrate, triacetin, tributyrin, diethyl carbonate, propylenecarbonate acetone, methyl ethyl ketone, dimethyl sulfoxide dimethylsulfone, tetrahydrofuran, capralactum, N,N-diethyl-m-toluamide,1-dodecylazacycloheptan-2-one,1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone and glycerol formalto form an injectable polymer solution. The dispersion may be introducedinto the infarct region of the heart where the solvent may dissipate andthe polymer may precipitate out of the dispersion to structurallyreinforce the infarct regional wall. In one embodiment, the disclosedpolymers may be used in any combination as co-polymers of two or morepolymers introduced to the infarct region.

Another possible reinforcing agent may include the use of a vinylpolymer and acrylate biocompatible polymer system. Once injected into aninfarct zone, the vinyl polymer/acrylate agent contacts water and thepolymer precipitates thus reinforcing the surrounding tissue of theinfarct region. In one embodiment, the vinyl polymer/acrylate agentincludes the following such as polyvinyl butyral, PBMA-HEMA, PEMA-HEMA,PMMA-HEMA and other acrylate copolymers that dissolve in ethanol,acetone and I-PA. In another embodiment, the vinyl polymer/acrylateagent introduced to the infarct region may be EVAL™ that has a solidphase or melt phase forming process. EVAL™ Resins have a highcrystalline structure. Thermoforming grades of EVAL™ resins havemonoclinic crystalline structure while most polyolefins have either ahexagonal or orthorhombic type structure. This characteristic providesflexibility within its thermoforming capabilities. In anotherembodiment, the vinyl polymer/acrylate agent introduced to the infarctregion may be BUTVAR™ (polyvinyl butyral). In one embodiment, the agentmay be P(BMA co-MMA) (Aldrich Chem.) in Diglyme. In another embodiment,the agent may be EVAL™, a co-polymer of ethylene and vinyl alcohol (EVALCo. of America, Houston, Tex.) in dimethyl acetamide. In anotherembodiment, the polymer may be PLGA (poly(lactide co-glycolide)(Birmingham Polymers, Birmingham, Ala.) in Diglyme.

Other components may act as a substrate for endogenous collagendeposition and protect the precipitated or remaining microparticles fromerosion. As the reinforcing gel degrades, the highly stable and smoothmicroparticles may be exposed to the fibroblast cell populationoccupying the site. This triggers the production of collagen to replacethe decomposing gel. Therefore, the infarct zone may be reinforced bythe collagen replacement of the temporary gel. The dispersed materialincludes the following group of microparticle materials: PMMA, P(MMA-coBMA), carbon microparticles (Durasphere), poly styrene, cross-linkedacrylic hydrogels and PLGA. In another embodiment, the cross-linkedacrylic hydrogel may include the following for example HEMA, AA, AMPS,acrylamide, N, N, di-methyl acrylamide, diacetone acrylamide, styrenesulfonate, and di or tri functional monomers. The di or tri-functionalmonomers may be EGDMA and DVB. Another example of durable microparticlesincludes pyrolytic carbon-coated microparticles. One example ofpyrolytic carbon-coated microparticles was originally produced forurinary incontinence (Carbon Medical Technologies) and trisacryl gelatinmicroparticles for use as embolization particles (Biosphere). Inaddition, the use of highly crystalline (and hydrolysis resistant) PLGAmicroparticles may outlast the carrier gel and also provide a usefulsubstrate for collagen deposition.

One or more contrast agents 1540 and/or suitable treatment agent(s) 1550may accompany the previously detailed components as a treatment of theinfarct region. The contrast agent or treatment agent may be conjugatedto or dissolved into the structural component prior to introduction tothe infarct area. The contrast agents may be used for detection in X-rayor MR analysis. The agents that may accompany the reinforcingcomponent(s) may include but are not limited to angiogenic agents, ACEinhibitors, angiotensin receptor blockers, SRCA pump (sarcoplasmicreticulum calcium pump) increasing agents, phospholamban inhibitors andanti-apoptotic drugs. These agents may be in the form of smallmolecules, peptides, proteins or gene products. The small molecules maybe optionally conjugated to a component of the solution, dispersed insolution, or dissolved in solution to improve the adhesion of thereinforcing components to the tissue. One embodiment is to conjugate apeptide with a conserved region that mediates adhesion processes.Another embodiment includes the use of a specific peptide conjugate witha RGD (arginine-glycine-asparagine) motif in the presence of thereinforcing component. In further embodiments, the RGD motif peptide mayinclude von Willebrand factor, osteopontin, fibronectin, fibrinogen,vitronectin, laminin and collagen.

In one embodiment, any of the described agents may be introduced in oneor more doses in a volume of about 1 μL to 1 mL. In another embodiment,any of the described agents may be introduced in one or more doses in avolume of about 1 μL to 300 μL. In another embodiment, any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 100 μL. In a preferred embodiment, the any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 50 μL.

Additionally, any one of these agents may be accompanied by one or morecontrast agent and/or suitable agent(s) for treatment of the region. Thecontrast agent or treatment agent may be conjugated to or dissolved intothe structural component prior to introduction to the infarct area. Theagents that may accompany the reinforcing component(s) may include butare not limited to angiogenic agents, ACE inhibitors, angiotensinreceptor blockers, SRCA pump increasing agents, phospholamban inhibitorsand anti-apoptotic drugs. These agents may be in the form of smallmolecules, peptides, proteins or gene products. The small molecules maybe optionally conjugated to a component of the solution, dispersed insolution, or dissolved in solution to improve the adhesion of thereinforcing components to the tissue. One embodiment is to conjugate apeptide with a conserved region that mediates adhesion processes.Another embodiment includes the use of a specific peptide conjugate witha RGD (arginine-glycine-aspartic acid) motif in the presence of thereinforcing component. In further embodiments, the RGD motif peptidecomprises the following: von Willebrand factor, osteopontin,fibronectin, fibrinogen, vitronectin, laminin or collagen.

Prevention of Myocardial Edema and “Cementing” of the Infarct Region

One of the initial responses of the process post-MI is myocardial edema.The edema is composed of extravasated blood evident within a few hoursafter infarction. This is followed by its dissolution within the nextfew hours. The process that occurs immediately post-MI is that theinfarct regional wall thickens and then it thins. The present inventionintroduces one or more clotting factors to the region thereby“cementing” the now clotted blood to reinforce the wall and thicken thewall. One method to clot the blood may use a dual solution technique. Inone embodiment, the first solution includes calcium chloride andthrombin and the second solution may include fibrinogen and transexamicacid. Transexamic acid is an anti-fibrinolytic agent. The introductionof these two solutions to the infarct region sequentially result inlocalized clotting of the blood that forms a structurally reinforcingmass within the region preventing thinning of the infarct site. Inanother embodiment, intra-venous pressure perfusion may be used todeliver the clot inducing solutions to the infarct zone. This preventsthe possibility of the clot releasing into the arterial circulation.Another possible component for promoting clotting may be the use ofshear-activated platelet fraction to induce localized clotting. Thisplatelet fraction may be isolated from the MI subject's own blood oranother source. Other factors encompass factors that are termedintrinsic and extrinsic factors. Intrinsic factors initiate clotting inthe absence of injury. Extrinsic factors initiate clotting that iscaused by injury. In one embodiment, the clotting factor used to ceasemyocardial edema and reinforce the ventricular wall at the infarct zonemay comprise the following: von Willebrand Factor (vWF), High MolecularWeight Kininogen (HMWK), Fibrinogen, Prothrombin, or Tissue FactorsIII-X. In another embodiment of the present invention, any combinationof the clotting factors mentioned previously may be used that mayprovide increased tensile strength the infarct regional wall.

Matrix Metalloproteinase Inhibitors Use in the Infarct Region

After an MI injury occurs macrophages tend to infiltrate the infarctregion. The macrophages release matrix metalloproteinases (MMPs). Asmembers of a zinc-containing endoproteinase family, the MMPs havestructural similarities but each enzyme has a different substratespecificity, produced by different cells and additionally have differentinducibilities. These enzymes cause destruction in the infarct zone. Oneimportant structural component destroyed by MMPs is the extracellularmatrix (ECM). The ECM is a complex structural entity surrounding andsupporting cells that are found within mammalian tissues. The ECM isoften referred to as the connective tissue. The ECM is composed of 3major classes of biomolecules: structural proteins, for example,collagen and elastin; specialized proteins, for example, fibrillin,fibronectin, and laminin; and proteoglycans. These are composed of aprotein core to which is attached long chains of repeating disaccharideunits termed of glycosaminoglycans (GAGs) forming extremely complex highmolecular weight components of the ECM. Collagen is the principalcomponent of the ECM and MMP induce ECM degradation and affect collagendeposition. Inhibitors of MMP(s) exist 1970 and some of these inhibitorsare tissue specific. It was previously demonstrated that acutepharmacological inhibition of MMPs or in some cases a deficiency inMMP-9 that the left ventricle dilatation is attenuated in the infarctheart of a mouse. See Creemers, E., et al., Matrix MetalloproteinaseInhibition After Myocardial Infarction, A New Approach to Prevent HeatFailure?, Circ. Res., Vol. 89, (2001), pp. 201-210. The inhibitors ofMMPs are referred to as tissue inhibitors of metalloproteinases (TIMPs).Synthetic forms of MMPIs also exist for example BB-94, AG3340, Ro32-355band GM 6001. It was previously shown that MMPIs reduce the remodeling inthe left ventricle by reducing wall thinning. These experiments wereperformed on rabbits. In addition, this study also demonstrated thatMMPI increases rather than decreases neovascularization in thesubendocardium. See Lindsey, et al., Selective matrix metalloproteinaseinhibitors reduce left ventricle remodeling but does not inhibitangiogenesis after myocardial infarction, Circulation, (Feb. 12, 2002),105(6):753-758. In the one embodiment MMPIs may be introduced to theinfarct region to delay the remodeling process by reducing the migrationof fibroblasts and deposition of collagen and prevent ECM degradation,reduce leukocyte influx and also reduce wall stress. In one embodiment,the MMPIs may include the following TIMPs including, but not limited to,TIMP-1, TIMP-2, TIMP-3 and TIMP-4 introduced to the infarct region incombination with introducing any of the described structurallyreinforcing agents to the infarct region. In another embodiment,naturally occurring inhibitors of MMPs may be increased by exogenousadministration of recombinant TIMPs. In another embodiment, the MMPIcomprises a synthetically derived MMPI introduced to the infarct regionin combination with the introduction of any of the describedstructurally reinforcing agents and/or applied stimulating devices(e.g., PG) to the infarct region. The introduction of MMPIs to theinfarct zone may be accomplished by several different methods. It iscritical that the introduction of these MMPI agents be accomplished by aminimally invasive technique. In one embodiment, MMPI agents will beintroduced to the region by a minimally invasive procedure to preventECM degradation. An agent or dispersion will be introduced in oneembodiment by multiple injections to the infarct region. This results inprevention of ECM degradation and increased strength to the regionalwall. In one embodiment, the MMPI agent may be injected in to theinfarct zone during an open chest procedure via a minimally invasiveprocedure. In another, the minimally invasive procedure may include oneof sub-xiphoid and percutaneous methods. In another embodiment, thepercutaneous introduction into the infarct zone may include one ofintra-ventricular needle, transvascular needle catheter and retrogradevenous perfusion. In addition, the MMPI agents may be introduced viasuspension or sustained release formula, for example, introduced inmicroparticles.

Structural Reinforcement of the Infarct Zone by Inducible Gel Systems

Photo-polymerizable hydrogels have been used before in tissueengineering applications. These gels are biocompatible and do not causethrombosis or tissue damage. These hydrogels may be photo-polymerized invivo and in vitro in the presence of ultraviolet (UV) or visible lightdepending on the photo initiation system. Photo-polymerizing materialsmay be spatially and temporally controlled by the polymerization rate.These hydrogels have very fast curing rates. A monomer or macromer formof the hydrogel may be introduced to the infarct zone for augmentationwith a photo initiator. Examples of these hydrogel materials include PEGacrylate derivatives, PEG methacrylate derivatives or modifiedpolysaccharides.

Visible light may be used to initiate interfacial photopolymerization ofa polyoxyethylene glycol (PEG)-co-poly(alpha-hydroxy acid) copolymerbased on PEG 8000 macromonomer in the presence of an initiator, forexample, Quantacure QTX. Initiator2-hydroxy-3-(3,4,dimethyl-9-oxo-9H-thioxanthen-2-yloxy)N,N,N-trimethyl-1-propaniumchloride photo-initiator may be obtained as Quantacure QTX. This is aspecific water-soluble photo-initiator that absorbs ultraviolet and/orvisible radiation and forms an excited state that may subsequently reactwith electron-donating sites and may produce free radicals. Thistechnology has been used to demonstrate adherence to porcine aortictissue, resulting in a hydrogel barrier that conformed to the region ofintroduction. The resulting matrix was optimized in vitro and resultedin the formation of a 5-100 microns thick barrier. See Lyman, M. D., etal., Characterization of the formation of interfacially photopolymerizedthin hydrogels in contact with arterial tissue Biomaterials, February1996, pp. 359-64, 17(3). Scaffolding may be directed to only the desiredarea of the ventricle using minimally invasive procedures discussedpreviously. The structural reinforcement could remain in place until itis cleared or degrade.

One embodiment includes introduction to the infarct zone of benzoinderivatives, hydroxalkylphenones, benziketals and acetophenonederivatives or similar compounds. These photo-initiators form radicalsupon exposure to UV light by either photocleavage or by hydrogenabstraction to initiate the reaction. The source of the UV or visiblelight may be supplied by means of a catheter, for example, a fiber optictip catheter or lead on a catheter, or, transdermally as described. Acatheter assembly may be used to deliver a light sensitive material. Thecatheter is designed to provide a delivery device with at least onelumen for one or more agent(s) and a light source for modification ofthe delivered agent. The catheter controller may house a switch for thelight source and a controller for agent deliver. In another embodiment,the photo-initiator Camphorquinone may be used. Camphorquinone has beenused extensively in dental applications and has a λ_(max) of 467nanometers. For example, this agent can be activated by a GaN blue LEDon the tip of a catheter. One embodiment includes the use of visiblelight at the end of the delivery catheter to induce the polymerizationevent in the presence of a light sensitive initiator. Another embodimentincludes the use of the photoinitiator, Camphorquinone that mayfacilitate the cross-linking of the hydrogel by a light on the tip of acatheter within the infarct region. Another embodiment includes the useof the photoinitiator, Quanticare QTX that may facilitate thecross-linking of the hydrogel by a light on the tip of a catheter withinthe infarct region. Another embodiment includes the use of a catheterwith a UVA light source to induce the polymerization event in thepresence of a light sensitive initiator. Other initiators ofpolymerization in the visible group include water soluble free radicalinitiator2-hydroxy-3-(3,4,dimethyl-9-oxo-9H-thioxanthen-2-yloxy)N,N,N-trimethyl-1-propaniumchloride. This cascade of events provides the necessary environment forinitiation of polymerization of suitable vinyl monomers or pre-polymersin aqueous form within the infarct region. See Kinart, et al.,Electrochemical studies of2-hydroxy-3-(3,4-dimethyl-9-oxo-9H-thioxanthen-2-yloxy)N,N,N-trimethyl-1-propaniumchloride, J. Electroanal. Chem., 1990, pp. 293-297, 294).

In one embodiment the photo-polymerizable material may be introduced tothe infarct regions during an open chest procedure or via a minimallyinvasive procedure. In another embodiment, the minimally invasiveprocedure includes sub-xiphoid and percutaneous methods. In anotherembodiment, the percutaneous introduction into the infarct zone mayinclude one of the following: intra-ventricular needle; transvascularneedle catheter; and retrograde venous perfusion. Any of theseembodiments may include the use of electrical stimulation via leads forpulse generation in and/or around the infarct region. A single boreneedle catheter may be used to introduce the photo-polymerizablematerial into the infarct zone. Once the agent is introduced to theregion, several heartbeats clear the excess agent into the ventricle andthis excess agent is cleared from the cardiac region. Once the excessmaterial is cleared, the light source may be introduced to inducepolymerization. Thus, the structural reinforcement is confined to thelocal area of damage where tissue augmentation is required. Thescaffolding may be made up of a resistant material or a biodegradablematerial. Some examples of biodegradable materials include PEG-co-poly(α-hydroxy acid) diacrylate macromers, derivatives of this material thatvary the length and composition of the α-hydroxy acid segment in theco-polymer, polypropylene fumarate-co-ethylene glycol and hyaluronicacid derivatives. The degradation rates of the polymers may be variedaccording to the optimum length of time the material is required toremain in the infarct region. It has been shown that the degradationrates of theses gels can be modified by the appropriate choice of theoligo(α-hydroxy acid) from as little as less than one day to as long as4 months. See Sawhney, A. S., et al., Bioerodable Hydrogels Based onPhotopolymerized Poly(ethylene glycol)-co-poly(a-hydroxyacid)+Diacrylate Macromers, Macromolecules. 1993, pp. 581-587, 26. Anyof these polymer chains may be formed in the presence of aphotoinitiator, such as Quantacure QTX, and a light source.

In one embodiment, any of the described agents may be introduced in oneor more doses in a volume of about 1 μL to 1 mL. In another embodiment,any of the described agents may be introduced in one or more doses in avolume of about 1 μL to 300 μL. In another embodiment, any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 100 μL. In a preferred embodiment, the any of thedescribed agents may be introduced in one or more doses in a volume ofabout 1 μL to 50 μL.

One way to introduce dual components to an infarct region is by using acatheter device. This avoids the possibility of injecting the agentsinto the exact same site. The delivery device of U.S. patent applicationSer. No. 10/414,602, incorporated herein, may be used to deliver thecomponents to the infarct region. The infarction is a region between theendocardium and the epicardium. The device is advanced to a site and thefirst component is delivered by extending a first needle. The componentis thereby dispersed in the infarct area. Then, this first needle isretracted while the second needle is extended. The second component isdispersed. The delivery of the two components to the area may be capableof forming a gel, or, may be two separate components.

FIG. 9A-9E illustrates the introduction of several components to theinfarct region 910 to treat an MI. One embodiment may be theintroduction of a first component 920, for example, microparticlesharboring a growth factor 940. Then, a second component 930 such ascells or a structurally reinforcing agent, is introduced through a dualbore needle (FIGS. 9C and 9D 950). FIG. 9E illustrates a schematic ofthe release of growth factor from the degrading microparticles 925.Optionally, a cellular component such as a knockout porcine heart cellmay also be introduced to the area. The thiol-containing component 905may be used to decrease the rate of decomposition of the scaffold andcontrol release of the fibroblast recruiting components of themicroparticles. A further optimal treatment includes the use ofelectrical stimulation around the infarct region as described above inconjunction with any combination of other components shown in FIG.9A-9E.

Ventricular Plugs

Another method for reinforcing the damaged wall of a ventricle mayinclude introduction of a solid material to the damaged area. The solidmaterial may be used to fill or bulk the region by introducing plugs ofthe solid material to the site and may increase the compliance of theventricle. These materials may be made of organic or silicon-basedpolymers, biodegradable polymers, non-biodegradable polymers, engineeredbiomaterials and/or metals. In one embodiment the plug may have barbs orpointed ends in order to lodge the material into the area and ensure itremains there. In other embodiments, the sealant or plug may add bulk tothe thinning wall of an infarct post myocardial infarction. This mayresult in an increase in the modulus of elasticity of the damaged area.In other embodiments, the sealant or plug may invoke an inflammatoryresponse to the infarct region. The inflammatory response will result inthe increase in angiogenic response capable of causing recruitment andactivation of fibroblasts that deposit additional collagen to bulk thethinning infarct region and increase the modulus of elasticity of thisregion. Still, other embodiments include the addition of a plug to thedamaged region of a ventricle that may add strength to the wall and alsocause an inflammatory response to the region.

In one embodiment, the plug supplied to the damaged region of theventricle may include biocompatible organic components. In otherembodiments, the plug supplied to the damaged region of the ventriclemay include a biocompatible silicone-based polymer. In otherembodiments, the plug supplied to the damaged region of the ventriclemay include biocompatible biodegradable polymers for example PLGA,Poly(hydroxyvalerate) and poly ortho esters etc. In other embodiments,the plug supplied to the damaged region of the ventricle may includebiocompatible non-biodegradable material for example polypropylene andPMMA. In still further embodiments, the plug supplied to the damagedregion of the ventricle may include biocompatible metal compounds, forexample, 316L, Co—Cr alloy, tantalum and titanium. Another advantage tousing a plug directly implanted in the region of interest may be to addadditional surface components to the plug such as side groups. Theseside groups may contain reactive side groups that react with exogenouslysupplied or endogenous collagen, for example type I and type IIIcollagen. Since collagen contains a significant number of lysine andhydroxyproline residues, these residues harbor primary amine andhydroxyl groups capable of reacting with other moieties. In oneembodiment, the plug supplied to the damaged region of the ventricle mayinclude surface aldehyde groups capable of reacting with the primaryamines of lysine in collagen.

The size and the shape of the plugs may vary depending on the situation.For example, polymeric plugs mentioned previously may be machined,injection molded, extruded or solution cast. In one embodiment, theshape of the plug may be elongated and thin in order to facilitatedelivery by a catheter device. These plugs may also possess a barb orside protrusion to prevent the plug from slipping out of the site onceit is introduced to the damaged region of the ventricle. In otherembodiments, the plug may be created in the shape similar to a screw ora helix. In one embodiment, the plug may be a polymeric material. Inother embodiments, the plug may be a polymeric material with SS anchorsfor example, a plug with a stainless steel band with anchors forembedding the plug into the site of interest. The size of the plug mayalso vary. In one embodiment, the radial diameter of the plug may befrom about 0.1 mm to about 5 mm. In other embodiments, the radialdiameter of the plug may be about 0.2 mm to about 3 mm. In otherembodiments, the length of the plug may be from about 1 mm to about 20mm. In other embodiments, the length of the plug may be about 2 mm toabout 12 mm. In addition to the size and shape of the plug, the numberof plugs supplied to a region in the ventricle may also vary dependingon the extent of damage and the condition of the subject. In oneembodiment, the number of plugs supplied to the region may about 1 toabout 200. In other embodiments, the number of plugs supplied to theregion may be about 5 to about 50. In still further embodiments, thenumber of plugs supplied to the region may be about 2 to about 20.

In one embodiment, the plug may be a processed biocompatiblebiomaterial. This biomaterial may be advantageous for recruiting cellsto the damaged region for additional strength to the site. One exampleof a biomaterial includes porcine derived Small Intestine Submucosa,termed SIS. This engineered biomaterial may be supplied from DePuy Incand the Cook Group. It is available in sterile sheets. SIS includes thecomplete small intestinal sub-mucosa, including de-cellularizedextracellular matrix (ECM) in a native configuration. It also includesimportant endogenous growth factors adhered to the matrix. SIS haspreviously been shown to recruit pluripotent bone marrow derived stemcells that adhere to the SIS and induce healing. SIS has previously beenused to repair rotator cuff injuries, diabetic foot ulcers and hipjoints. SIS has been shown to re-absorb after a period of approximately3 to 4 months. After re-absorption, the healed live tissue has replacedthe matrix. In one embodiment, small disks of SIS may be supplied to aregion in the ventricle for example an infarct region. The SIS disks mayprovide similar recruitment of cells into the damaged myocardium. Thesecells may then transform into viable muscle tissue and may formcontractile myocytes. In another embodiment a processed biocompatiblebiomaterial (e.g., SIS) may be used for structural reinforcement of aninfarct region in combination with electrical stimulation such asimplanting leads for pulsing the site to unload the infarct area before,during and/or after the structural component is introduced.

There are several methods that may be used to introduce any of the plugsdescribed. An optimum approach for introduction of the plugs mayinclude, but is not limited, to introduction to the infarct regionand/or the border zone of an infarct region during an open-heartprocedure; or through a minimally invasive procedure, for example,sub-xiphoid or percutaneous methods, for example, with anintra-ventricular catheter or transvascular catheter (venous orarterial). One embodiment for introducing the plugs to the infarctregion may include directly introducing the plugs to the site during anopen-heart surgical procedure.

One or more contrast agents and/or suitable treatment agent(s) mayaccompany the previously detailed components. The contrast agent ortreatment agent may be dispersed into, conjugated to, or dissolved intothe plug component prior to introduction to the infarct area. Thecontrast agents may be used for detection in X-ray or MR analysis. Theagents that may accompany the reinforcing component(s) may include butare not limited to angiogenic agents, ACE inhibitors, angiotensinreceptor blockers, SRCA pump (sarcoplasmic reticulum calcium pump)increasing agents, phospholamban inhibitors and anti-apoptotic drugs.These agents may be in the form of small molecules, peptides, proteinsor gene products. The agents may be optionally conjugated to a componentof the resin mix that makes a plug, dispersed in a plug solution priorto forming a plug, or dissolved in a plug solution prior to forming aplug, or packed into machined pockets or reservoirs in a plug to elicita biological effect (e.g., improve implant adhesion, recruit cells,promote healing). One embodiment is to conjugate a peptide with aconserved region that mediates adhesion processes. Another embodimentincludes the use of a specific peptide conjugate with a RGD(arginine-glycine-aspartic acid) motif or the peptide receptor to RGD,such as DDM (aspartate-aspartate-methionine) in the presence of thereinforcing component. In further embodiments, the RGD motif peptide mayinclude von Willebrand factor, osteopontin, fibronectin, fibrinogen,vitronectin, laminin or collagen.

In the foregoing specification, the embodiments have been described withreference to specific exemplary embodiments. It will, however, beevident that various modifications and changes may be made withoutdeparting from the broader spirit and scope of the invention as detailedin the appended claims. The specification and drawings are, accordingly,to be regarded in an illustrative rather than a restrictive sense.

EXAMPLES Example 1

In one example a 2-component gel may be injected via a dual needlecatheter to an infarct region. One possible 2-component gel material mayinclude Na-Alginate (component 1) which will likely ionically crosslink(often within seconds) when added to a soluble solution of calcium,barium and/or strontium (component 2). One potential crosslinker for thecomponents may be CaCl₂ (calcium chloride). In one example, covalentlyconjugating a peptide or protein to some of the acid groups of thealginate may enhance a cellular response. For example, conjugation ofRGD groups or gelatin may promote cell adhesion, since these are bindingsites for cells. The amine (N) terminus of gelatin or RGD may beconjugated to the acid groups of alginate via carbodiimide chemistry,forming an amide. The reaction may be mediated to higher yield withfewer side products (such as the inactive N-acyl urea) by first formingan active ester with, for example, 1-hydroxybenzotriazole or N-hydroxysuccinimide, before adding the peptide or protein. Side products andunreacted material may be removed by dialysis.

When used as an adjunct to cellular injection, the cells may be firstmixed with the alginate or alginate-peptide conjugate. This mix isinjected down one needle lumen, followed by an injection or simultaneouswith an injection of calcium chloride solution. The gel may prevent thecells from migrating, but is sufficiently porous to allow for transportof nutrients and waste products. Concentrations and volumes: 1% Alginate(Protanal LF10/60, FMC Biopolymers)-2 parts, 3.2% Calcium Chloridedihydrate in water-1 part.

Example 2

In one example, a person with an mild or severe desynchrony (such asleft bundle branch block), detectable through QRS duration,echocardiography, or other means, and with a history of a previousmyocardial infarction, may first receive a catheter delivered injectionof micronized porcine urinary bladder matrix (UBM) particulate(cryogenically ground UBM and resuspended at 5% weight/volume in PBS) toachieve alterations in the mechanical properties of the previous/scarredregion. Following injection(s), implantation of a cardiac rhythmmanagement device may be used to provide cardiac resynchronizationtherapy (CRT) to alter the desynchronized condition and alter thepatient's progression to heart failure.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it areapparent to those of skill in the art that variations maybe applied tothe COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itare apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A method to minimize post-infarct ventricularremodeling, comprising: delivering electrical stimulation using one ormore sensing channels for sensing intrinsic cardiac activity selectedfrom the group consisting of: (i) a plurality of pacing channels fordelivering pacing pulses; (ii) a controller for controlling the deliveryof pacing pulses in accordance with a pacing algorithm; and (iii) apatch having a plurality of pacing electrodes incorporated into thepacing channels; and delivering at least one structurally reinforcingcomponent to the infarct region.
 2. The method of claim 1, furthercomprising, a drug delivery system for delivering an agent selected froma group consisting of an ACE inhibitor, a beta blocker, a growth factor,and an anti-apoptotic factor.
 3. The method of claim 1, furthercomprising, an impedance sensor for detecting changes in wall motion andwall thickness in an area in proximity to the infarct and wherein thecontroller is programmed to modify the delivery of pacing pulses.
 4. Themethod of claim 1 wherein delivering at least one structurallyreinforcing agent increases the modulus of elasticity of the infarctregion.
 5. The method of claim 1 wherein delivering at least onestructurally reinforcing agent to the infarct region comprisesdelivering microspheres.
 6. The method of claim 1 wherein thestructurally reinforcing agent comprises a modified and/or unmodifiedalginate gel material.